Antisense nucleic acids

ABSTRACT

The present invention provides an oligomer which efficiently enables to cause skipping of the 53rd exon in the human dystrophin gene. Also provided is a pharmaceutical composition which causes skipping of the 53rd exon in the human dystrophin gene with a high efficiency.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of copending application Ser. No.16/449,537 (allowed), filed Jun. 24, 2019, which is a Continuation ofapplication Ser. No. 15/619,996, filed Jun. 12, 2017 (now U.S. Pat. No.10,329,319 issued Jun. 25, 2019), which is a Continuation of applicationSer. No. 14/615,504, filed Feb. 6, 2015 (now U.S. Pat. No. 9,708,361issued Jul. 18, 2017), which is a Continuation of application Ser. No.13/819,520, filed Apr. 10, 2013 (now U.S. Pat. No. 9,079,934 issued Jul.14, 2015), which is a PCT National Stage of PCT/JP2011/070318 filed Aug.31, 2011, which claims priority to JP Application No. 2010-196032 filedSep. 1, 2010, all of which are incorporated by reference in theirentireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 9, 2019 isnamed 209658_0001_09_US_594705_ST25.txt and is 24,765 bytes in size.

TECHNICAL FIELD

The present invention relates to an antisense oligomer which causesskipping of exon 53 in the human dystrophin gene, and a pharmaceuticalcomposition comprising the oligomer.

BACKGROUND ART

Duchenne muscular dystrophy (DMD) is the most frequent form ofhereditary progressive muscular dystrophy that affects one in about3,500 newborn boys. Although the motor functions are rarely differentfrom healthy humans in infancy and childhood, muscle weakness isobserved in children from around 4 to 5 years old. Then, muscle weaknessprogresses to the loss of ambulation by about 12 years old and death dueto cardiac or respiratory insufficiency in the twenties. DMD is such asevere disorder. At present, there is no effective therapy for DMDavailable, and it has been strongly desired to develop a noveltherapeutic agent.

DMD is known to be caused by a mutation in the dystrophin gene. Thedystrophin gene is located on X chromosome and is a huge gene consistingof 2.2 million DNA nucleotide pairs. DNA is transcribed into mRNAprecursors, and introns are removed by splicing to synthesize mRNA inwhich 79 exons are joined together. This mRNA is translated into 3,685amino acids to produce the dystrophin protein. The dystrophin protein isassociated with the maintenance of membrane stability in muscle cellsand necessary to make muscle cells less fragile. The dystrophin genefrom patients with DMD contains a mutation and hence, the dystrophinprotein, which is functional in muscle cells, is rarely expressed.Therefore, the structure of muscle cells cannot be maintained in thebody of the patients with DMD, leading to a large influx of calcium ionsinto muscle cells. Consequently, an inflammation-like response occurs topromote fibrosis so that muscle cells can be regenerated only withdifficulty.

Becker muscular dystrophy (BMD) is also caused by a mutation in thedystrophin gene. The symptoms involve muscle weakness accompanied byatrophy of muscle but are typically mild and slow in the progress ofmuscle weakness, when compared to DMD. In many cases, its onset is inadulthood. Differences in clinical symptoms between DMD and BMD areconsidered to reside in whether the reading frame for amino acids on thetranslation of dystrophin mRNA into the dystrophin protein is disruptedby the mutation or not (Non-Patent Document 1). More specifically, inDMD, the presence of mutation shifts the amino acid reading frame sothat the expression of functional dystrophin protein is abolished,whereas in BMD the dystrophin protein that functions, thoughimperfectly, is produced because the amino acid reading frame ispreserved, while a part of the exons are deleted by the mutation.

Exon skipping is expected to serve as a method for treating DMD. Thismethod involves modifying splicing to restore the amino acid readingframe of dystrophin mRNA and induce expression of the dystrophin proteinhaving the function partially restored (Non-Patent Document 2). Theamino acid sequence part, which is a target for exon skipping, will belost. For this reason, the dystrophin protein expressed by thistreatment becomes shorter than normal one but since the amino acidreading frame is maintained, the function to stabilize muscle cells ispartially retained. Consequently, it is expected that exon skipping willlead DMD to the similar symptoms to that of BMD which is milder. Theexon skipping approach has passed the animal tests using mice or dogsand now is currently assessed in clinical trials on human DMD patients.

The skipping of an exon can be induced by binding of antisense nucleicacids targeting either 5′ or 3′ splice site or both sites, orexon-internal sites. An exon will only be included in the mRNA when bothsplice sites thereof are recognized by the spliceosome complex. Thus,exon skipping can be induced by targeting the splice sites withantisense nucleic acids. Furthermore, the binding of an SR protein to anexonic splicing enhancer (ESE) is considered necessary for an exon to berecognized by the splicing mechanism. Accordingly, exon skipping canalso be induced by targeting ESE.

Since a mutation of the dystrophin gene may vary depending on DMDpatients, antisense nucleic acids need to be designed based on the siteor type of respective genetic mutation. In the past, antisense nucleicacids that induce exon skipping for all 79 exons were produced by SteveWilton, et al., University of Western Australia (Non-Patent Document 3),and the antisense nucleic acids which induce exon skipping for 39 exonswere produced by Annemieke Aartsma-Rus, et al., Netherlands (Non-PatentDocument 4).

It is considered that approximately 8% of all DMD patients may betreated by skipping the 53rd exon (hereinafter referred to as “exon53”). In recent years, a plurality of research organizations reported onthe studies where exon 53 in the dystrophin gene was targeted for exonskipping (Patent Documents 1 to 4; Non-Patent Document 5). However, atechnique for skipping exon 53 with a high efficiency has not yet beenestablished.

-   Patent Document 1: International Publication WO 2006/000057-   Patent Document 2: International Publication WO 2004/048570-   Patent Document 3: US 2010/0168212-   Patent Document 4: International Publication WO 2010/048586-   Non-Patent Document 1: Monaco A. P. et al., Genomics 1988; 2: p.    90-95-   Non-Patent Document 2: Matsuo M., Brain Dev 1996; 18: p. 167-172-   Non-Patent Document 3: Wilton S. D., e t al., Molecular Therapy    2007: 15: p. 1288-96-   Non-Patent Document 4: Annemieke Aartsma-Rus et al., (2002)    Neuromuscular Disorders 12: S71-S77-   Non-Patent Document 5: Linda J. Popplewell et al., (2010)    Neuromuscular Disorders, vol. 20, no. 2, p. 102-10

DISCLOSURE OF THE INVENTION

Under the foregoing circumstances, antisense oligomers that stronglyinduce exon 53 skipping in the dystrophin gene and muscular dystrophytherapeutics comprising oligomers thereof have been desired.

As a result of detailed studies of the structure of the dystrophin gene,the present inventors have found that exon 53 skipping can be inducedwith a high efficiency by targeting the sequence consisting of the 32ndto the 56th nucleotides from the 5′ end of exon 53 in the mRNA precursor(hereinafter referred to as “pre-mRNA”) in the dystrophin gene withantisense oligomers. Based on this finding, the present inventors haveaccomplished the present invention.

That is, the present invention is as follows.

[1] An antisense oligomer which causes skipping of the 53rd exon in thehuman dystrophin gene, consisting of a nucleotide sequence complementaryto any one of the sequences consisting of the 31st to the 53rd, the 31stto the 54th, the 31st to the 55th, the 31st to the 56th, the 31st to the57th, the 31st to the 58th, the 32nd to the 53rd, the 32nd to the 54th,the 32nd to the 55th, the 32nd to the 56th, the 32nd to the 57th, the32nd to the 58th, the 33rd to the 53rd, the 33rd to the 54th, the 33rdto the 55th, the 33rd to the 56th, the 33rd to the 57th, the 33rd to the58th, the 34th to the 53rd, the 34th to the 54th, the 34th to the 55th,the 34th to the 56th, the 34th to the 57th, the 34th to the 58th, the35th to the 53rd, the 35th to the 54th, the 35th to the 55th, the 35thto the 56th, the 35th to the 57th, the 35th to the 58th, the 36th to the53rd, the 36th to the 54th, the 36th to the 55th, the 36th to the 56th,the 36th to the 57th, or the 36th to the 58th nucleotides, from the 5′end of the 53rd exon in the human dystrophin gene.

[2] The antisense oligomer according to [1] above, which is anoligonucleotide.

[3] The antisense oligomer according to [2] above, wherein the sugarmoiety and/or the phosphate-binding region of at least one nucleotideconstituting the oligonucleotide is modified.

[4] The antisense oligomer according to [3] above, wherein the sugarmoiety of at least one nucleotide constituting the oligonucleotide is aribose in which the 2′-OH group is replaced by any one selected from thegroup consisting of OR, R, R′OR, SH, SR, NH₂, NHR, NR₂, N₃, CN, F, Cl,Br and I (wherein R is an alkyl or an aryl and R′ is an alkylene).

[5] The antisense oligomer according to [3] or [4] above, wherein thephosphate-binding region of at least one nucleotide constituting theoligonucleotide is any one selected from the group consisting of aphosphorothioate bond, a phosphorodithioate bond, an alkylphosphonatebond, a phosphoramidate bond and a boranophosphate bond.

[6] The antisense oligomer according to [1] above, which is a morpholinooligomer.

[7] The antisense oligomer according to [6] above, which is aphosphorodiamidate morpholino oligomer.

[8] The antisense oligomer according to any one of [1] to [7] above,wherein the 5′ end is any one of the groups of chemical formulae (1) to(3) below:

[9] The antisense oligomer according to any one of [1] to [8] above,consisting of a nucleotide sequence complementary to the sequencesconsisting of the 32nd to the 56th or the 36th to the 56th nucleotidesfrom the 5′ end of the 53rd exon in the human dystrophin gene.

[10] The antisense oligomer according to any one of [1] to [8] above,consisting of the nucleotide sequence shown by any one selected from thegroup consisting of SEQ ID NOS: 2 to 37.

[11] The antisense oligomer according to any one of [1] to [8] above,consisting of the nucleotide sequence shown by any one selected from thegroup consisting of SEQ ID NOS: 11, 17, 23, 29 and 35.

[12] The antisense oligomer according to any one of [1] to [8] above,consisting of the nucleotide sequence shown by SEQ ID NO: 11 or 35.

[13] A pharmaceutical composition for the treatment of musculardystrophy, comprising as an active ingredient the antisense oligomeraccording to any one of [1] to [12] above, or a pharmaceuticallyacceptable salt or hydrate thereof.

The antisense oligomer of the present invention can induce exon 53skipping in the human dystrophin gene with a high efficiency. Inaddition, the symptoms of Duchenne muscular dystrophy can be effectivelyalleviated by administering the pharmaceutical composition of thepresent invention.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the efficiency of exon 53 skipping in the human dystrophingene in human rhabdomyosarcoma cell line (RD cells).

FIG. 2 shows the efficiency of exon 53 skipping in the human dystrophingene in the cells where human myoD gene is introduced into human normaltissue-derived fibroblasts (TIG-119 cells) to induce differentiationinto muscle cells.

FIG. 3 shows the efficiency of exon 53 skipping in the human dystrophingene in the cells where human myoD gene is introduced into human DMDpatient-derived fibroblasts (5017 cells) to induce differentiation intomuscle cells.

FIG. 4 shows the efficiency of exon 53 skipping in the human dystrophingene in the cells where human myoD gene is introduced into fibroblastsfrom human DMD patient (with deletion of exons 45-52) to inducedifferentiation into muscle cells.

FIG. 5 shows the efficiency of exon 53 skipping in the human dystrophingene in the cells where human myoD gene is introduced into fibroblastsfrom human DMD patient (with deletion of exons 48-52) to inducedifferentiation into muscle cells.

FIG. 6 shows the efficiency of exon 53 skipping in the human dystrophingene in the cells where human myoD gene is introduced into fibroblastsfrom human DMD patient (with deletion of exons 48-52) to inducedifferentiation into muscle cells.

FIG. 7 shows the efficiency of exon 53 skipping in the human dystrophingene in the cells where human myoD gene is introduced into fibroblastsfrom human DMD patient (with deletion of exons 45-52 or deletion ofexons 48-52) to induce differentiation into muscle cells.

FIG. 8 shows the efficiency of exon 53 skipping in the human dystrophingene in the cells where human myoD gene is introduced into fibroblastsfrom human DMD patient (with deletion of exons 45-52) to inducedifferentiation into muscle cells.

FIG. 9 shows the efficiency of exon 53 skipping (2′-OMe-S-RNA) in thehuman dystrophin gene in human rhabdomyosarcoma cells (RD cells).

FIG. 10 shows the efficiency of exon 53 skipping (2′-OMe-S-RNA) in thehuman dystrophin gene in human rhabdomyosarcoma cells (RD cells).

FIG. 11 shows the efficiency of exon 53 skipping (2′-OMe-S-RNA) in thehuman dystrophin gene in human rhabdomyosarcoma cells (RD cells).

FIG. 12 shows the efficiency of exon 53 skipping (2′-OMe-S-RNA) in thehuman dystrophin gene in human rhabdomyosarcoma cells (RD cells).

FIG. 13 shows the efficiency of exon 53 skipping (2′-OMe-S-RNA) in thehuman dystrophin gene in human rhabdomyosarcoma cells (RD cells).

FIG. 14 shows the efficiency of exon 53 skipping (2′-OMe-S-RNA) in thehuman dystrophin gene in human rhabdomyosarcoma cells (RD cells).

FIG. 15 shows the efficiency of exon 53 skipping (2′-OMe-S-RNA) in thehuman dystrophin gene in human rhabdomyosarcoma cells (RD cells).

FIG. 16 shows the efficiency of exon 53 skipping (2′-OMe-S-RNA) in thehuman dystrophin gene in human rhabdomyosarcoma cells (RD cells).

FIG. 17 shows the efficiency of exon 53 skipping (2′-OMe-S-RNA) in thehuman dystrophin gene in human rhabdomyosarcoma cells (RD cells).

FIG. 18 shows the efficiency of exon 53 skipping in the human dystrophingene in human rhabdomyosarcoma cells (RD cells) at the respectiveconcentrations of the oligomers.

FIG. 19 shows the efficiency of exon 53 skipping in the human dystrophingene in human rhabdomyosarcoma cells (RD cells) at the respectiveconcentrations of the oligomers.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention is described in detail. Theembodiments described below are intended to be presented by way ofexample merely to describe the invention but not limited only to thefollowing embodiments. The present invention may be implemented invarious ways without departing from the gist of the invention.

All of the publications, published patent applications, patents andother patent documents cited in the specification are hereinincorporated by reference in their entirety. The specification herebyincorporates by reference the contents of the specification and drawingsin the Japanese Patent Application (No. 2010-196032) filed Sep. 1, 2010,from which the priority was claimed.

1. Antisense Oligomer

The present invention provides the antisense oligomer (hereinafterreferred to as the “oligomer of the present invention”) which causesskipping of the 53rd exon in the human dystrophin gene, consisting of anucleotide sequence complementary to any one of the sequences(hereinafter also referred to as “target sequences”) consisting of the31st to the 53rd, the 31st to the 54th, the 31st to the 55th, the 31stto the 56th, the 31st to the 57th, the 31st to the 58th, the 32nd to the53rd, the 32nd to the 54th, the 32nd to the 55th, the 32nd to the 56th,the 32nd to the 57th, the 32nd to the 58th, the 33rd to the 53rd, the33rd to the 54th, the 33rd to the 55th, the 33rd to the 56th, the 33rdto the 57th, the 33rd to the 58th, the 34th to the 53rd, the 34th to the54th, the 34th to the 55th, the 34th to the 56th, the 34th to the 57th,the 34th to the 58th, the 35th to the 53rd, the 35th to the 54th, the35th to the 55th, the 35th to the 56th, the 35th to the 57th, the 35thto the 58th, the 36th to the 53rd, the 36th to the 54th, the 36th to the55th, the 36th to the 56th, the 36th to the 57th, or the 36th to the58th nucleotides, from the 5′ end of the 53rd exon in the humandystrophin gene.

[Exon 53 in Human Dystrophin Gene]

In the present invention, the term “gene” is intended to mean a genomicgene and also include cDNA, mRNA precursor and mRNA. Preferably, thegene is mRNA precursor, i.e., pre-mRNA.

In the human genome, the human dystrophin gene locates at locus Xp21.2.The human dystrophin gene has a size of 3.0 Mbp and is the largest geneamong known human genes. However, the coding regions of the humandystrophin gene are only 14 kb, distributed as 79 exons throughout thehuman dystrophin gene (Roberts, R G., et al., Genomics, 16: 536-538(1993)). The pre-mRNA, which is the transcript of the human dystrophingene, undergoes splicing to generate mature mRNA of 14 kb. Thenucleotide sequence of human wild-type dystrophin gene is known (GenBankAccession No. NM_004006).

The nucleotide sequence of exon 53 in the human wild-type dystrophingene is represented by SEQ ID NO: 1.

The oligomer of the present invention is designed to cause skipping ofexon 53 in the human dystrophin gene, thereby modifying the proteinencoded by DMD type of dystrophin gene into the BMD type of dystrophinprotein. Accordingly, exon 53 in the dystrophin gene that is the targetof exon skipping by the oligomer of the present invention includes bothwild and mutant types.

Specifically, exon 53 mutants of the human dystrophin gene include thepolynucleotides defined in (a) or (b) below.

(a) A polynucleotide that hybridizes under stringent conditions to apolynucleotide consisting of a nucleotide sequence complementary to thenucleotide sequence of SEQ ID NO: 1; and,

(b) A polynucleotide consisting of a nucleotide sequence having at least90% identity with the nucleotide sequence of SEQ ID NO: 1.

As used herein, the term “polynucleotide” is intended to mean DNA orRNA.

As used herein, the term “polynucleotide that hybridizes under stringentconditions” refers to, for example, a polynucleotide obtained by colonyhybridization, plaque hybridization, Southern hybridization or the like,using as a probe all or part of a polynucleotide consisting of anucleotide sequence complementary to the nucleotide sequence of, e.g.,SEQ ID NO: 1. The hybridization method which may be used includesmethods described in, for example, “Sambrook & Russell, MolecularCloning: A Laboratory Manual Vol. 3, Cold Spring Harbor, LaboratoryPress 2001,” “Ausubel, Current Protocols in Molecular Biology, JohnWiley & Sons 1987-1997,” etc.

As used herein, the term “complementary nucleotide sequence” is notlimited only to nucleotide sequences that form Watson-Crick pairs withtarget nucleotide sequences, but is intended to also include nucleotidesequences which form Wobble base pairs. As used herein, the termWatson-Crick pair refers to a pair of nucleobases in which hydrogenbonds are formed between adenine-thymine, adenine-uracil orguanine-cytosine, and the term Wobble base pair refers to a pair ofnucleobases in which hydrogen bonds are formed between guanine-uracil,inosine-uracil, inosine-adenine or inosine-cytosine. As used herein, theterm “complementary nucleotide sequence” does not only refers to anucleotide sequence 100% complementary to the target nucleotide sequencebut also refers to a complementary nucleotide sequence that may contain,for example, 1 to 3, 1 or 2, or one nucleotide non-complementary to thetarget nucleotide sequence.

As used herein, the term “stringent conditions” may be any of lowstringent conditions, moderate stringent conditions or high stringentconditions. The term “low stringent conditions” are, for example, 5×SSC,5×Denhardt's solution, 0.5% SDS, 50% formamide at 32° C. The term“moderate stringent conditions” are, for example, 5×SSC, 5×Denhardt'ssolution, 0.5% SDS, 50% formamide at 42° C., or 5×SSC, 1% SDS, 50 mMTris-HCl (pH 7.5), 50% formamide at 42° C. The term “high stringentconditions” are, for example, 5×SSC, 5×Denhardt's solution, 0.5% SDS,50% formamide at 50° C. or 0.2×SSC, 0.1% SDS at 65° C. Under theseconditions, polynucleotides with higher homology are expected to beobtained efficiently at higher temperatures, although multiple factorsare involved in hybridization stringency including temperature, probeconcentration, probe length, ionic strength, time, salt concentrationand others, and those skilled in the art may appropriately select thesefactors to achieve similar stringency.

When commercially available kits are used for hybridization, forexample, an Alkphos Direct Labeling and Detection System (GE Healthcare)may be used. In this case, according to the attached protocol, aftercultivation with a labeled probe overnight, the membrane is washed witha primary wash buffer containing 0.1% (w/v) SDS at 55° C., therebydetecting hybridized polynucleotides. Alternatively, in producing aprobe based on the entire or part of the nucleotide sequencecomplementary to the nucleotide sequence of SEQ ID NO: 1, hybridizationcan be detected with a DIG Nucleic Acid Detection Kit (RocheDiagnostics) when the probe is labeled with digoxigenin (DIG) using acommercially available reagent (e.g., a PCR Labeling Mix (RocheDiagnostics), etc.).

In addition to the polynucleotides described above, otherpolynucleotides that can be hybridized include polynucleotides having90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% orhigher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99%or higher, 99.1% or higher, 99.2% or higher, 99.3% or higher, 99.4% orhigher, 99.5% or higher, 99.6% or higher, 99.7% or higher, 99.8% orhigher or 99.9% or higher identity with the polynucleotide of SEQ ID NO:1, as calculated by homology search software BLAST using the defaultparameters.

The identity between nucleotide sequences may be determined usingalgorithm BLAST (Basic Local Alignment Search Tool) by Karlin andAltschul (Proc. Natl. Acad. Sci. USA 872264-2268, 1990; Proc. Natl.Acad. Sci. USA 90: 5873, 1993). Programs called BLASTN and BLASTX basedon the BLAST algorithm have been developed (Altschul S F, et al: J. Mol.Biol. 215: 403, 1990). When a nucleotide sequence is sequenced usingBLASTN, the parameters are, for example, score=100 and wordlength=12.When BLAST and Gapped BLAST programs are used, the default parametersfor each program are employed.

Examples of the nucleotide sequences complementary to the sequencesconsisting of the 31st to the 53rd, the 31st to the 54th, the 31st tothe 55th, the 31st to the 56th, the 31st to the 57th, the 31st to the58th, the 32nd to the 53rd, the 32nd to the 54th, the 32nd to the 55th,the 32nd to the 56th, the 32nd to the 57th, the 32nd to the 58th, the33rd to the 53rd, the 33rd to the 54th, the 33rd to the 55th, the 33rdto the 56th, the 33rd to the 57th, the 33rd to the 58th, the 34th to the53rd, the 34th to the 54th, the 34th to the 55th, the 34th to the 56th,the 34th to the 57th, the 34th to the 58th, the 35th to the 53rd, the35th to the 54th, the 35th to the 55th, the 35th to the 56th, the 35thto the 57th, the 35th to the 58th, the 36th to the 53rd, the 36th to the54th, the 36th to the 55th, the 36th to the 56th, the 36th to the 57thand the 36th to the 58th nucleotides, from the 5′ end of exon 53.

TABLE 1 Target se- quence in exon SEQ ID 53Complementary nucleotide sequence NO: 31-535′-CCGGTTCTGAAGGTGTTCTTGTA-3′ SEQ ID NO: 2 31-545′-TCCGGTTCTGAAGGTGTTCTTGTA-3′ SEQ ID NO: 3 31-555′-CTCCGGTTCTGAAGGTGTTCTTGTA-3′ SEQ ID NO: 4 31-565′-CCTCCGGTTCTGAAGGTGTTCTTGTA-3′ SEQ ID NO: 5 31-575′-GCCTCCGGTTCTGAAGGTGTTCTTGTA-3′ SEQ ID NO: 6 31-585′-TGCCTCCGGTTCTGAAGGTGTTCTTGTA-3′ SEQ ID NO: 7 32-535′-CCGGTTCTGAAGGTGTTCTTGT-3′ SEQ ID NO: 8 32-545′-TCCGGTTCTGAAGGTGTTCTTGT-3′ SEQ ID NO: 9 32-555′-CTCCGGTTCTGAAGGTGTTCTTGT-3′ SEQ ID NO: 10 32-565′-CCTCCGGTTCTGAAGGTGTTCTTGT-3′ SEQ ID NO: 11 32-575′-GCCTCCGGTTCTGAAGGTGTTCTTGT-3′ SEQ ID NO: 12 32-585′-TGCCTCCGGTTCTGAAGGTGTTCTTGT-3′ SEQ ID NO: 13 33-535′-CCGGTTCTGAAGGTGTTCTTG-3′ SEQ ID NO: 14 33-545′-TCCGGTTCTGAAGGTGTTCTTG-3′ SEQ ID NO: 15 33-555′-CTCCGGTTCTGAAGGTGTTTCTTG-3′ SEQ ID NO: 16 33-565′-CCTCCGGTTCTGAAGGTGTTCTTG-3′ SEQ ID NO: 17 33-575′-GCCTCCGGTTCTGAAGGTGTTCTTG-3′ SEQ ID NO: 18 33-585′-TGCCTCCGGTTCTGAAGGTGTTCTTG-3′ SEQ ID NO: 19 34-535′-CCGGTTCTGAAGGTGTTCTT-3′ SEQ ID NO: 20 34-545′-TCCGGTTCTGAAGGTGTTCTT-3′ SEQ ID NO: 21 34-555′-CTCCGGTTCTGAAGGTGTTCTT-3′ SEQ ID NO: 22 34-565′-CCTCCGGTTCTGAAGGTGTTCTT-3′ SEQ ID NO: 23 34-575′-GCCTCCGGTTCTGAAGGTGTTCTT-3′ SEQ ID NO: 24 34-585′-TGCCTCCGGTTCTGAAGGTGTTCTT-3′ SEQ ID NO: 25 35-535′-CCGGTTCTGAAGGTGTTCT-3′ SEQ ID NO: 26 35-54 5′-TCCGGTTCTGAAGGTGTTCT-3′SEQ ID NO: 27 35-55 5′-CTCCGGTTCTGAAGGTGTTCT-3′ SEQ ID NO: 28 35-565′-CCTCCGGTTCTGAAGGTGTTCT-3′ SEQ ID NO: 29 35-575′-GCCTCCGGTTCTGAAGGTGTTCT-3′ SEQ ID NO: 30 35-585′-TGCCTCCGGTTCTGAAGGTGTTCT-3′ SEQ ID NO: 31 36-535′-CCGGTTCTGAAGGTGTTC-3′ SEQ ID NO: 32 36-54 5′-TCCGGTTCTGAAGGTGTTC-3′SEQ ID NO: 33 36-55 5′-CTCCGGTTCTGAAGGTGTTC-3′ SEQ ID NO: 34 36-565′-CCTCCGGTTCTGAAGGTGTTC-3′ SEQ ID NO: 35 36-575′-GCCTCCGGTTCTGAAGGTGTTC-3′ SEQ ID NO: 36 36-585′-TGCCTCCGGTTCTGAAGGTGTTC-3′ SEQ ID NO: 37

It is preferred that the oligomer of the present invention consists of anucleotide sequence complementary to any one of the sequences consistingof the 32nd to the 56th, the 33rd to the 56th, the 34th to the 56th, the35th to the 56th or the 36th to the 56th nucleotides (e.g., SEQ ID NO:11, SEQ ID NO: 17, SEQ ID NO: 23, SEQ ID NO: 29 or SEQ ID NO: 35), fromthe 5′ end of the 53rd exon in the human dystrophin gene.

Preferably, the oligomer of the present invention consists of anucleotide sequence complementary to any one of the sequences consistingof the 32nd to the 56th or the 36th to the 56th nucleotides (e.g., SEQID NO: 11 or SEQ ID NO: 35), from the 5′ end of the 53rd exon in thehuman dystrophin gene.

The term “cause skipping of the 53rd exon in the human dystrophin gene”is intended to mean that by binding of the oligomer of the presentinvention to the site corresponding to exon 53 of the transcript (e.g.,pre-mRNA) of the human dystrophin gene, for example, the nucleotidesequence corresponding to the 5′ end of exon 54 is spliced at the 3′side of the nucleotide sequence corresponding to the 3′ end of exon 51in DMD patients with deletion of, exon 52 when the transcript undergoessplicing, thus resulting in formation of mature mRNA which is free ofcodon frame shift.

Accordingly, it is not required for the oligomer of the presentinvention to have a nucleotide sequence 100% complementary to the targetsequence, as far as it causes exon 53 skipping in the human dystrophingene. The oligomer of the present invention may include, for example, 1to 3, 1 or 2, or one nucleotide non-complementary to the targetsequence.

Herein, the term “binding” described above is intended to mean that whenthe oligomer of the present invention is mixed with the transcript ofhuman dystrophin gene, both are hybridized under physiologicalconditions to form a double strand nucleic acid. The term “underphysiological conditions” refers to conditions set to mimic the in vivoenvironment in terms of pH, salt composition and temperature. Theconditions are, for example, 25 to 40° C., preferably 37° C., pH 5 to 8,preferably pH 7.4 and 150 mM of sodium chloride concentration.

Whether the skipping of exon 53 in the human dystrophin gene is causedor not can be confirmed by introducing the oligomer of the presentinvention into a dystrophin expression cell (e.g., humanrhabdomyosarcoma cells), amplifying the region surrounding exon 53 ofmRNA of the human dystrophin gene from the total RNA of the dystrophinexpression cell by RT-PCR and performing nested PCR or sequence analysison the PCR amplified product.

The skipping efficiency can be determined as follows. The mRNA for thehuman dystrophin gene is collected from test cells; in the mRNA, thepolynucleotide level “A” of the band where exon 53 is skipped and thepolynucleotide level “B” of the band where exon 53 is not skipped aremeasured. Using these measurement values of “A” and “B,” the efficiencyis calculated by the following equation:Skipping efficiency (%)=A/(A+B)×100

The oligomer of the present invention includes, for example, anoligonucleotide, morpholino oligomer or peptide nucleic acid (PNA),having a length of 18 to 28 nucleotides. The length is preferably from21 to 25 nucleotides and morpholino oligomers are preferred.

The oligonucleotide described above (hereinafter referred to as “theoligonucleotide of the present invention”) is the oligomer of thepresent invention composed of nucleotides as constituent units. Suchnucleotides may be any of ribonucleotides, deoxyribonucleotides andmodified nucleotides.

The modified nucleotide refers to one having fully or partly modifiednucleobases, sugar moieties and/or phosphate-binding regions, whichconstitute the ribonucleotide or deoxyribonucleotide.

The nucleobase includes, for example, adenine, guanine, hypoxanthine,cytosine, thymine, uracil, and modified bases thereof. Examples of suchmodified nucleobases include, but not limited to, pseudouracil,3-methyluracil, dihydrouracil, 5-alkylcytosines (e.g.,5-methylcytosine), 5-alkyluracils (e.g., 5-ethyluracil), 5-halouracils(5-bromouracil), 6-azapyrimidine, 6-alkylpyrimidines (6-methyluracil),2-thiouracil, 4-thiouracil, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5′-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyluracil, 1-methyladenine, 1-methylhypoxanthine,2,2-dimethylguanine, 3-methylcytosine, 2-methyladenine, 2-methylguanine,N6-methyladenine, 7-methylguanine, 5-methoxyaminomethyl-2-thiouracil,5-methylaminomethyluracil, 5-methylcarbonylmethyluracil,5-methyloxyuracil, 5-methyl-2-thiouracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid,2-thiocytosine, purine, 2,6-diaminopurine, 2-aminopurine, isoguanine,indole, imidazole, xanthine, etc.

Modification of the sugar moiety may include, for example, modificationsat the 2′-position of ribose and modifications of the other positions ofthe sugar. The modification at the 2′-position of ribose includesreplacement of the 2′-OH of ribose with OR, R, R′OR, SH, SR, NH₂, NHR,NR₂, N₃, CN, F, Cl, Br or I, wherein R represents an alkyl or an aryland R′ represents an alkylene.

The modification for the other positions of the sugar includes, forexample, replacement of O at the 4′ position of ribose or deoxyribosewith S, bridging between 2′ and 4′ positions of the sugar, e.g., LNA(locked nucleic acid) or ENA (2′-O,4′-C-ethylene-bridged nucleic acids),but is not limited thereto.

A modification of the phosphate-binding region includes, for example, amodification of replacing phosphodiester bond with phosphorothioatebond, phosphorodithioate bond, alkyl phosphonate bond, phosphoroamidatebond or boranophosphate bond (Enya et al: Bioorganic & MedicinalChemistry, 2008, 18, 9154-9160) (cf., e.g., Japan DomesticRe-Publications of PCT Application Nos. 2006/129594 and 2006/038608).

The alkyl is preferably a straight or branched alkyl having 1 to 6carbon atoms. Specific examples include methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl,isopentyl, neopentyl, tert-pentyl, n-hexyl and isohexyl. The alkyl mayoptionally be substituted. Examples of such substituents are a halogen,an alkoxy, cyano and nitro. The alkyl may be substituted with 1 to 3substituents.

The cycloalkyl is preferably a cycloalkyl having 5 to 12 carbon atoms.Specific examples include cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl, cyclodecyl and cyclododecyl.

The halogen includes fluorine, chlorine, bromine and iodine.

The alkoxy is a straight or branched alkoxy having 1 to 6 carbon atomssuch as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy,sec-butoxy, tert-butoxy, n-pentyloxy, isopentyloxy, n-hexyloxy,isohexyloxy, etc. Among others, an alkoxy having 1 to 3 carbon atoms ispreferred.

The aryl is preferably an aryl having 6 to 10 carbon atoms. Specificexamples include phenyl, α-naphthyl and β-naphthyl. Among others, phenylis preferred. The aryl may optionally be substituted. Examples of suchsubstituents are an alkyl, a halogen, an alkoxy, cyano and nitro. Thearyl may be substituted with one to three of such substituents.

The alkylene is preferably a straight or branched alkylene having 1 to 6carbon atoms. Specific examples include methylene, ethylene,trimethylene, tetramethylene, pentamethylene, hexamethylene, 2-(ethyl)trimethylene and 1-(methyl) tetramethylene.

The acyl includes a straight or branched alkanoyl or aroyl. Examples ofthe alkanoyl include formyl, acetyl, 2-methylacetyl, 2,2-dimethylacetyl,propionyl, butyryl, isobutyryl, pentanoyl, 2,2-dimethylpropionyl,hexanoyl, etc. Examples of the aroyl include benzoyl, toluoyl andnaphthoyl. The aroyl may optionally be substituted at substitutablepositions and may be substituted with an alkyl(s).

Preferably, the oligonucleotide of the present invention is the oligomerof the present invention containing a constituent unit represented bygeneral formula below wherein the —OH group at position 2′ of ribose issubstituted with methoxy and the phosphate-binding region is aphosphorothioate bond:

wherein Base represents a nucleobase.

The oligonucleotide of the present invention may be easily synthesizedusing various automated synthesizer (e.g., AKTA oligopilot plus 10/100(GE Healthcare)). Alternatively, the synthesis may also be entrusted toa third-party organization (e.g., Promega Inc., or Takara Co.), etc.

The morpholino oligomer of the present invention is the oligomer of thepresent invention comprising the constituent unit represented by generalformula below:

wherein Base has the same significance as defined above, and,W represents a group shown by any one of the following groups:

wherein X represents —CH₂R¹, —O—CH₂R¹, —S—CH₂R¹, —NR₂R³ or F;

R¹ represents H or an alkyl;

R² and R³, which may be the same or different, each represents H, analkyl, a cycloalkyl or an aryl;

Y₁ represents O, S, CH₂ or NR¹;

Y₂ represents O, S or NR¹;

Z represents O or S.

Preferably, the morpholino oligomer is an oligomer comprising aconstituent unit represented by general formula below(phosphorodiamidate morpholino oligomer (hereinafter referred to as“PMO”)).

wherein Base, R² and R³ have the same significance as defined above.

The morpholino oligomer may be produced in accordance with, e.g., WO1991/009033 or WO 2009/064471. In particular, PMO can be produced by theprocedure described in WO 2009/064471 or produced by the process shownbelow.

[Method for Producing PMO]

An embodiment of PMO is, for example, the compound represented bygeneral formula (I) below (hereinafter PMO (I)).

wherein Base, R² and R³ have the same significance as defined above;and,

n is a given integer of 1 to 99, preferably a given integer of 18 to 28.

PMO (I) can be produced in accordance with a known method, for example,can be produced by performing the procedures in the following steps.

The compounds and reagents used in the steps below are not particularlylimited so long as they are commonly used to prepare PMO.

Also, the following steps can all be carried out by the liquid phasemethod or the solid phase method (using manuals or commerciallyavailable solid phase automated synthesizers). In producing PMO by thesolid phase method, it is desired to use automated synthesizers in viewof simple operation procedures and accurate synthesis.

(1) Step A:

The compound represented by general formula (II) below (hereinafterreferred to as Compound (II)) is reacted with an acid to prepare thecompound represented by general formula (III) below (hereinafterreferred to as Compound (III)):

wherein n, R² and R³ have the same significance as defined above;each B^(P) independently represents a nucleobase which may optionally beprotected;T represents trityl, monomethoxytrityl or dimethoxytrityl; and,L represents hydrogen, an acyl or a group represented by general formula(IV) below (hereinafter referred to as group (IV)).

The “nucleobase” for B^(P) includes the same “nucleobase” as in Base,provided that the amino or hydroxy group in the nucleobase shown byB^(P) may be protected.

Such protective group for amino is not particularly limited so long asit is used as a protective group for nucleic acids. Specific examplesinclude benzoyl, 4-methoxybenzoyl, acetyl, propionyl, butyryl,isobutyryl, phenylacetyl, phenoxyacetyl, 4-tert-butylphenoxyacetyl,4-isopropylphenoxyacetyl and (dimethylamino)methylene. Specific examplesof the protective group for the hydroxy group include 2-cyanoethyl,4-nitrophenethyl, phenylsulfonylethyl, methylsulfonylethyl andtrimethylsilylethyl, and phenyl, which may be substituted by 1 to 5electron-withdrawing group at optional substitutable positions,diphenylcarbamoyl, dimethylcarbamoyl, diethylcarbamoyl,methylphenylcarbamoyl, 1-pyrolidinylcarbamoyl, morpholinocarbamoyl,4-(tert-butylcarboxy) benzyl, 4-[(dimethylamino)carboxy]benzyl and4-(phenylcarboxy)benzyl, (cf., e.g., WO 2009/064471).

The “solid carrier” is not particularly limited so long as it is acarrier usable for the solid phase reaction of nucleic acids. It isdesired for the solid carrier to have the following properties: e.g.,(i) it is sparingly soluble in reagents that can be used for thesynthesis of morpholino nucleic acid derivatives (e.g., dichloromethane,acetonitrile, tetrazole, N-methylimidazole, pyridine, acetic anhydride,lutidine, trifluoroacetic acid); (ii) it is chemically stable to thereagents usable for the synthesis of morpholino nucleic acidderivatives; (iii) it can be chemically modified; (iv) it can be chargedwith desired morpholino nucleic acid derivatives; (v) it has a strengthsufficient to withstand high pressure through treatments; and (vi) ithas a uniform particle diameter range and distribution. Specifically,swellable polystyrene (e.g., aminomethyl polystyrene resin 1%dibenzylbenzene crosslinked (200-400 mesh) (2.4-3.0 mmol/g)(manufactured by Tokyo Chemical Industry), Aminomethylated PolystyreneResin.HCl [dibenzylbenzene 1%, 100-200 mesh] (manufactured by PeptideInstitute, Inc.)), non-swellable polystyrene (e.g., Primer Support(manufactured by GE Healthcare)), PEG chain-attached polystyrene (e.g.,NH₂-PEG resin (manufactured by Watanabe Chemical Co.), TentaGel resin),controlled pore glass (controlled pore glass; CPG) (manufactured by,e.g., CPG), oxalyl-controlled pore glass (cf., e.g., Alul et al.,Nucleic Acids Research, Vol. 19, 1527 (1991)), TentaGelsupport-aminopolyethylene glycol-derivatized support (e.g., Wright etal., cf., Tetrahedron Letters, Vol. 34, 3373 (1993)), and a copolymer ofPoros-polystyrene/divinylbenzene.

A “linker” which can be used is a known linker generally used to connectnucleic acids or morpholino nucleic acid derivatives. Examples include3-aminopropyl, succinyl, 2,2′-diethanolsulfonyl and a long chain alkylamino (LCAA).

This step can be performed by reacting Compound (11) with an acid.

The “acid” which can be used in this step includes, for example,trifluoroacetic acid, dichloroacetic acid and trichloroacetic acid. Theacid used is appropriately in a range of, for example, 0.1 molequivalent to 1000 mol equivalents based on 1 mol of Compound (II),preferably in a range of 1 mol equivalent to 100 mol equivalents basedon 1 mol of Compound (II).

An organic amine can be used in combination with the acid describedabove. The organic amine is not particularly limited and includes, forexample, triethylamine. The amount of the organic amine used isappropriately in a range of, e.g., 0.01 mol equivalent to 10 molequivalents, and preferably in a range of 0.1 mol equivalent to 2 molequivalents, based on 1 mol of the acid.

When a salt or mixture of the acid and the organic amine is used in thisstep, the salt or mixture includes, for example, a salt or mixture oftrifluoroacetic acid and triethylamine, and more specifically, a mixtureof 1 equivalent of triethylamine and 2 equivalents of trifluoroaceticacid.

The acid which can be used in this step may also be used in the form ofa dilution with an appropriate solvent in a concentration of 0.1% to30%. The solvent is not particularly limited as far as it is inert tothe reaction, and includes, for example, dichloromethane, acetonitrile,an alcohol (ethanol, isopropanol, trifluoroethanol, etc.), water, or amixture thereof.

The reaction temperature in the reaction described above is preferablyin a range of, e.g., 10° C. to 50° C., more preferably, in a range of20° C. to 40° C., and most preferably, in a range of 25° C. to 35° C.

The reaction time may vary depending upon kind of the acid used andreaction temperature, and is appropriately in a range of 0.1 minute to24 hours in general, and preferably in a range of 1 minute to 5 hours.

After completion of this step, a base may be added, if necessary, toneutralize the acid remained in the system. The “base” is notparticularly limited and includes, for example, diisopropylamine. Thebase may also be used in the form of a dilution with an appropriatesolvent in a concentration of 0.1% (v/v) to 30% (v/v).

The solvent used in this step is not particularly limited so long as itis inert to the reaction, and includes dichloromethane, acetonitrile, analcohol (ethanol, isopropanol, trifluoroethanol, etc.), water, and amixture thereof. The reaction temperature is preferably in a range of,e.g., 10° C. to 50° C., more preferably, in a range of 20° C. to 40° C.,and most preferably, in a range of 25° C. to 35° C.

The reaction time may vary depending upon kind of the base used andreaction temperature, and is appropriately in a range of 0.1 minute to24 hours in general, and preferably in a range of 1 minute to 5 hours.

In Compound (II), the compound of general formula (IIa) below(hereinafter Compound (IIa)), wherein n is 1 and L is a group (IV), canbe produced by the following procedure.

wherein B^(P), T, linker and solid carrier have the same significance asdefined above.Step 1:

The compound represented by general formula (V) below is reacted with anacylating agent to prepare the compound represented by general formula(VI) below (hereinafter referred to as Compound (VI)).

wherein B^(P), T and linker have the same significance as defined above;and,R⁴ represents hydroxy, a halogen or amino.

This step can be carried out by known procedures for introducinglinkers, using Compound (V) as the starting material.

In particular, the compound represented by general formula (VIa) belowcan be produced by performing the method known as esterification, usingCompound (V) and succinic anhydride.

wherein B^(P) and T have the same significance as defined above.Step 2:

Compound (VI) is reacted with a solid career by a condensing agent toprepare Compound (IIa).

wherein B^(P), R⁴, T, linker and solid carrier have the samesignificance as defined above.

This step can be performed using Compound (VI) and a solid carrier inaccordance with a process known as condensation reaction.

In Compound (II), the compound represented by general formula (IIa2)below wherein n is 2 to 99 and L is a group represented by generalformula (IV) can be produced by using Compound (IIa) as the startingmaterial and repeating step A and step B of the PMO production methoddescribed in the specification for a desired number of times.

wherein B^(P), R², R³, T, linker and solid carrier have the samesignificance as defined above; and,n′ represents 1 to 98.

In Compound (II), the compound of general formula (IIb) below wherein nis 1 and L is hydrogen can be produced by the procedure described in,e.g., WO 1991/009033.

wherein B^(P) and T have the same significance as defined above.

In Compound (II), the compound represented by general formula (IIb2)below wherein n is 2 to 99 and L is hydrogen can be produced by usingCompound (IIb) as the starting material and repeating step A and step Bof the PMO production method described in the specification for adesired number of times.

wherein B^(P), n′, R², R³ and T have the same significance as definedabove.

In Compound (II), the compound represented by general formula (IIc)below wherein n is 1 and L is an acyl can be produced by performing theprocedure known as acylation reaction, using Compound (IIb).

wherein B^(P) and T have the same significance as defined above; and,R⁵ represents an acyl.

In Compound (II), the compound represented by general formula (IIc2)below wherein n is 2 to 99 and L is an acyl can be produced by usingCompound (IIc) as the starting material and repeating step A and step Bof the PMO production method described in the specification for adesired number of times.

wherein B^(P), n′, R², R³, R⁵ and T have the same significance asdefined above.(2) Step B

Compound (III) is reacted with a morpholino monomer compound in thepresence of a base to prepare the compound represented by generalformula (VII) below (hereinafter referred to as Compound (VII)):

wherein B^(P), L, n, R², R³ and T have the same significance as definedabove.

This step can be performed by reacting Compound (III) with themorpholino monomer compound in the presence of a base.

The morpholino monomer compound includes, for example, compoundsrepresented by general formula (VIII) below:

wherein B^(P), R², R³ and T have the same significance as defined above.

The “base” which can be used in this step includes, for example,diisopropylamine, triethylamine and N-ethylmorpholine. The amount of thebase used is appropriately in a range of 1 mol equivalent to 1000 molequivalents based on 1 mol of Compound (III), preferably, 10 molequivalents to 100 mol equivalents based on 1 mol of Compound (III).

The morpholino monomer compound and base which can be used in this stepmay also be used as a dilution with an appropriate solvent in aconcentration of 0.1% to 30%. The solvent is not particularly limited asfar as it is inert to the reaction, and includes, for example,N,N-dimethylimidazolidone, N-methylpiperidone, DMF, dichloromethane,acetonitrile, tetrahydrofuran, or a mixture thereof.

The reaction temperature is preferably in a range of, e.g., 0° C. to100° C., and more preferably, in a range of 10° C. to 50° C.

The reaction time may vary depending upon kind of the base used andreaction temperature, and is appropriately in a range of 1 minute to 48hours in general, and preferably in a range of 30 minutes to 24 hours.

Furthermore, after completion of this step, an acylating agent can beadded, if necessary. The “acylating agent” includes, for example, aceticanhydride, acetyl chloride and phenoxyacetic anhydride. The acylatingagent may also be used as a dilution with an appropriate solvent in aconcentration of 0.1% to 30%. The solvent is not particularly limited asfar as it is inert to the reaction, and includes, for example,dichloromethane, acetonitrile, an alcohol(s) (ethanol, isopropanol,trifluoroethanol, etc.), water, or a mixture thereof.

If necessary, a base such as pyridine, lutidine, collidine,triethylamine, diisopropylethylamine, N-ethylmorpholine, etc. may alsobe used in combination with the acylating agent. The amount of theacylating agent is appropriately in a range of 0.1 mol equivalent to10000 mol equivalents, and preferably in a range of 1 mol equivalent to1000 mol equivalents. The amount of the base is appropriately in a rangeof, e.g., 0.1 mol equivalent to 100 mol equivalents, and preferably in arange of 1 mol equivalent to 10 mol equivalents, based on 1 mol of theacylating agent.

The reaction temperature in this reaction is preferably in a range of10° C. to 50° C., more preferably, in a range of 10° C. to 50° C., muchmore preferably, in a range of 20° C. to 40° C., and most preferably, ina range of 25° C. to 35° C. The reaction time may vary depending uponkind of the acylating agent used and reaction temperature, and isappropriately in a range of 0.1 minute to 24 hours in general, andpreferably in a range of 1 minute to 5 hours.

(3) Step C:

In Compound (VII) produced in Step B, the protective group is removedusing a deprotecting agent to prepare the compound represented bygeneral formula (IX).

wherein Base, B^(P), L, n, R², R³ and T have the same significance asdefined above.

This step can be performed by reacting Compound (VII) with adeprotecting agent.

The “deprotecting agent” includes, e.g., conc. ammonia water andmethylamine. The “deprotecting agent” used in this step may also be usedas a dilution with, e.g., water, methanol, ethanol, isopropyl alcohol,acetonitrile, tetrahydrofuran, DMF, N,N-dimethylimidazolidone,N-methylpiperidone, or a mixture of these solvents. Among others,ethanol is preferred. The amount of the deprotecting agent used isappropriately in a range of, e.g., 1 mol equivalent to 100000 molequivalents, and preferably in a range of 10 mol equivalents to 1000 molequivalents, based on 1 mol of Compound (VII).

The reaction temperature is appropriately in a range of 15° C. to 75°C., preferably, in a range of 40° C. to 70° C., and more preferably, ina range of 50° C. to 60° C. The reaction time for deprotection may varydepending upon kind of Compound (VII), reaction temperature, etc., andis appropriately in a range of 10 minutes to 30 hours, preferably 30minutes to 24 hours, and more preferably in a range of 5 hours to 20hours.

(4) Step D:

PMO (I) is produced by reacting Compound (IX) produced in step C with anacid:

wherein Base, n, R², R³ and T have the same significance as definedabove.

This step can be performed by adding an acid to Compound (IX).

The “acid” which can be used in this step includes, for example,trichloroacetic acid, dichloroacetic acid, acetic acid, phosphoric acid,hydrochloric acid, etc. The acid used is appropriately used to allow thesolution to have a pH range of 0.1 to 4.0, and more preferably, in arange of pH 1.0 to 3.0. The solvent is not particularly limited so longas it is inert to the reaction, and includes, for example, acetonitrile,water, or a mixture of these solvents thereof.

The reaction temperature is appropriately in a range of 10° C. to 50°C., preferably, in a range of 20° C. to 40° C., and more preferably, ina range of 25° C. to 35° C. The reaction time for deprotection may varydepending upon kind of Compound (IX), reaction temperature, etc., and isappropriately in a range of 0.1 minute to 5 hours, preferably 1 minuteto 1 hour, and more preferably in a range of 1 minute to 30 minutes.

PMO (I) can be obtained by subjecting the reaction mixture obtained inthis step to conventional means of separation and purification such asextraction, concentration, neutralization, filtration, centrifugalseparation, recrystallization, reversed phase column chromatography C₈to C₁₈, cation exchange column chromatography, anion exchange columnchromatography, gel filtration column chromatography, high performanceliquid chromatography, dialysis, ultrafiltration, etc., alone or incombination thereof. Thus, the desired PMO (I) can be isolated andpurified (cf., e.g., WO 1991/09033).

In purification of PMO (I) using reversed phase chromatography, e.g., asolution mixture of 20 mM triethylamine/acetate buffer and acetonitrilecan be used as an elution solvent.

In purification of PMO (I) using ion exchange chromatography, e.g., asolution mixture of 1 M saline solution and 10 mM sodium hydroxideaqueous solution can be used as an elution solvent.

A peptide nucleic acid is the oligomer of the present invention having agroup represented by the following general formula as the constituentunit:

wherein Base has the same significance as defined above.

Peptide nucleic acids can be prepared by referring to, e.g., thefollowing literatures.

-   1) P. E. Nielsen, M. Egholm, R. H. Berg, O. Buchardt, Science, 254,    1497 (1991)-   2) M. Egholm, O. Buchardt, P. E. Nielsen, R. H. Berg, Jacs., 114,    1895 (1992)-   3) K. L. Dueholm, M. Egholm, C. Behrens, L. Christensen, H. F.    Hansen, T. Vulpius, K. H. Petersen, R. H. Berg, P. E. Nielsen, O.    Buchardt, J. Org. Chem., 59, 5767 (1994)-   4) L. Christensen, R. Fitzpatrick, B. Gildea, K. H. Petersen, H. E    Hansen, T. Koch, M. Egholm, O. Buchardt, P. E. Nielsen, J.    Coull, R. H. Berg, J. Pept. Sci., 1, 175 (1995)-   5) T. Koch, H. F. Hansen, P. Andersen, T. Larsen, H. G Batz, K.    Otteson, H. Orum, J. Pept. Res., 49, 80 (1997)

In the oligomer of the present invention, the 5′ end may be any ofchemical structures (1) to (3) below, and preferably is (3)-OH.

Hereinafter, the groups shown by (1), (2) and (3) above are referred toas “Group (1),” “Group (2)” and “Group (3),” respectively.

2. Pharmaceutical Composition

The oligomer of the present invention causes exon 53 skipping with ahigher efficiency as compared to the prior art antisense oligomers. Itis thus expected that conditions of muscular dystrophy can be relievedwith high efficience by administering the pharmaceutical compositioncomprising the oligomer of the present invention to DMD patients. Forexample, when the pharmaceutical composition comprising the oligomer ofthe present invention is used, the same therapeutic effects can beachieved even in a smaller dose than that of the oligomers of the priorart. Accordingly, side effects can be alleviated and such is economical.

In another embodiment, the present invention provides the pharmaceuticalcomposition for the treatment of muscular dystrophy, comprising as anactive ingredient the oligomer of the present invention, apharmaceutically acceptable salt or hydrate thereof (hereinafterreferred to as “the composition of the present invention”).

Examples of the pharmaceutically acceptable salt of the oligomer of thepresent invention contained in the composition of the present inventionare alkali metal salts such as salts of sodium, potassium and lithium;alkaline earth metal salts such as salts of calcium and magnesium; metalsalts such as salts of aluminum, iron, zinc, copper, nickel, cobalt,etc.; ammonium salts; organic amine salts such as salts of t-octylamine,dibenzylamine, morpholine, glucosamine, phenylglycine alkyl ester,ethylenediamine, N-methylglucamine, guanidine, diethylamine,triethylamine, dicyclohexylamine, N,N′-dibenzylethylenediamine,chloroprocaine, procaine, diethanolamine, N-benzylphenethylamine,piperazine, tetramethylammonium, tris(hydroxymethyl)aminomethane;hydrohalide salts such as salts of hydrofluorates, hydrochlorides,hydrobromides and hydroiodides; inorganic acid salts such as nitrates,perchlorates, sulfates, phosphates, etc.; lower alkane sulfonates suchas methanesulfonates, trifluoromethanesulfonates and ethanesulfonates;arylsulfonates such as benzenesulfonates and p-toluenesulfonates;organic acid salts such as acetates, malates, fumarates, succinates,citrates, tartarates, oxalates, maleates, etc.; and, amino acid saltssuch as salts of glycine, lysine, arginine, ornithine, glutamic acid andaspartic acid. These salts may be produced by known methods.Alternatively, the oligomer of the present invention contained in thecomposition of the present invention may be in the form of a hydratethereof.

Administration route for the composition of the present invention is notparticularly limited so long as it is pharmaceutically acceptable routefor administration, and can be chosen depending upon method oftreatment. In view of easiness in delivery to muscle tissues, preferredare intravenous administration, intraarterial administration,intramuscular administration, subcutaneous administration, oraladministration, tissue administration, transdermal administration, etc.Also, dosage forms which are available for the composition of thepresent invention are not particularly limited, and include, forexample, various injections, oral agents, drips, inhalations, ointments,lotions, etc.

In administration of the oligomer of the present invention to patientswith muscular dystrophy, the composition of the present inventionpreferably contains a carrier to promote delivery of the oligomer tomuscle tissues. Such a carrier is not particularly limited as far as itis pharmaceutically acceptable, and examples include cationic carrierssuch as cationic liposomes, cationic polymers, etc., or carriers usingviral envelope. The cationic liposomes are, for example, liposomescomposed of 2-O-(2-diethylaminoethyl)carabamoyl-1,3-O-dioleoylglyceroland phospholipids as the essential constituents (hereinafter referred toas “liposome A”), Oligofectamine (registered trademark) (manufactured byInvitrogen Corp.), Lipofectin (registered trademark) (manufactured byInvitrogen Corp.), Lipofectamine (registered trademark) (manufactured byInvitrogen Corp.), Lipofectamine 2000 (registered trademark)(manufactured by Invitrogen Corp.), DMRIE-C (registered trademark)(manufactured by Invitrogen Corp.), GeneSilencer (registered trademark)(manufactured by Gene Therapy Systems), TransMessenger (registeredtrademark) (manufactured by QIAGEN, Inc.), TransIT TKO (registeredtrademark) (manufactured by Mirus) and Nucleofector II (Lonza). Amongothers, liposome A is preferred. Examples of cationic polymers are JetSI(registered trademark) (manufactured by Qbiogene, Inc.) and Jet-PEI(registered trademark) (polyethylenimine, manufactured by Qbiogene,Inc.). An example of carriers using viral envelop is GenomeOne(registered trademark) (HVJ-E liposome, manufactured by IshiharaSangyo). Alternatively, the medical devices described in Japanese PatentNo. 2924179 and the cationic carriers described in Japanese DomesticRe-Publication PCT Nos. 2006/129594 and 2008/096690 may be used as well.

A concentration of the oligomer of the present invention contained inthe composition of the present invention may vary depending on kind ofthe carrier, etc., and is appropriately in a range of 0.1 nM to 100 μM,preferably in a range of 1 nM to 10 μM, and more preferably in a rangeof 10 nM to 1 μM. A weight ratio of the oligomer of the presentinvention contained in the composition of the present invention and thecarrier (carrier/oligomer of the present invention) may vary dependingon property of the oligomer, type of the carrier, etc., and isappropriately in a range of 0.1 to 100, preferably in a range of 1 to50, and more preferably in a range of 10 to 20.

In addition to the oligomer of the present invention and the carrierdescribed above, pharmaceutically acceptable additives may also beoptionally formulated in the composition of the present invention.Examples of such additives are emulsification aids (e.g., fatty acidshaving 6 to 22 carbon atoms and their pharmaceutically acceptable salts,albumin and dextran), stabilizers (e.g., cholesterol and phosphatidicacid), isotonizing agents (e.g., sodium chloride, glucose, maltose,lactose, sucrose, trehalose), and pH controlling agents (e.g.,hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, sodiumhydroxide, potassium hydroxide and triethanolamine). One or more ofthese additives can be used. The content of the additive in thecomposition of the present invention is appropriately 90 wt % or less,preferably 70 wt % or less and more preferably, 50 wt % or less.

The composition of the present invention can be prepared by adding theoligomer of the present invention to a carrier dispersion and adequatelystirring the mixture. Additives may be added at an appropriate stepeither before or after addition of the oligomer of the presentinvention. An aqueous solvent that can be used in adding the oligomer ofthe present invention is not particularly limited as far as it ispharmaceutically acceptable, and examples are injectable water orinjectable distilled water, electrolyte fluid such as physiologicalsaline, etc., and sugar fluid such as glucose fluid, maltose fluid, etc.A person skilled in the art can appropriately choose conditions for pHand temperature for such matter.

The composition of the present invention may be prepared into, e.g., aliquid form and its lyophilized preparation. The lyophilized preparationcan be prepared by lyophilizing the composition of the present inventionin a liquid form in a conventional manner. The lyophilization can beperformed, for example, by appropriately sterilizing the composition ofthe present invention in a liquid form, dispensing an aliquot into avial container, performing preliminary freezing for 2 hours atconditions of about −40 to −20° C., performing a primary drying at 0 to10° C. under reduced pressure, and then performing a secondary drying atabout 15 to 25° C. under reduced pressure. In general, the lyophilizedpreparation of the composition of the present invention can be obtainedby replacing the content of the vial with nitrogen gas and capping.

The lyophilized preparation of the composition of the present inventioncan be used in general upon reconstitution by adding an optionalsuitable solution (reconstitution liquid) and redissolving thepreparation. Such a reconstitution liquid includes injectable water,physiological saline and other infusion fluids. A volume of thereconstitution liquid may vary depending on the intended use, etc., isnot particularly limited, and is suitably 0.5 to 2-fold greater than thevolume prior to lyophilization or no more than 500 mL.

It is desired to control a dose of the composition of the presentinvention to be administered, by taking the following factors intoaccount: the type and dosage form of the oligomer of the presentinvention contained; patients' conditions including age, body weight,etc.; administration route; and the characteristics and extent of thedisease. A daily dose calculated as the amount of the oligomer of thepresent invention is generally in a range of 0.1 mg to 10 g/human, andpreferably 1 mg to 1 g/human. This numerical range may vary occasionallydepending on type of the target disease, administration route and targetmolecule. Therefore, a dose lower than the range may be sufficient insome occasion and conversely, a dose higher than the range may berequired occasionally. The composition can be administered from once toseveral times daily or at intervals from one day to several days.

In still another embodiment of the composition of the present invention,there is provided a pharmaceutical composition comprising a vectorcapable of expressing the oligonucleotide of the present invention andthe carrier described above. Such an expression vector may be a vectorcapable of expressing a plurality of the oligonucleotides of the presentinvention. The composition may be formulated with pharmaceuticallyacceptable additives as in the case with the composition of the presentinvention containing the oligomer of the present invention. Aconcentration of the expression vector contained in the composition mayvary depending upon type of the career, etc., and is appropriately in arange of 0.1 nM to 100 μM, preferably in a range of 1 nM to 10 μM, andmore preferably in a range of 10 nM to 1 μM. A weight ratio of theexpression vector contained in the composition and the carrier(carrier/expression vector) may vary depending on property of theexpression vector, type of the carrier, etc., and is appropriately in arange of 0.1 to 100, preferably in a range of 1 to 50, and morepreferably in a range of 10 to 20. The content of the carrier containedin the composition is the same as in the case with the composition ofthe present invention containing the oligomer of the present invention,and a method for producing the same is also the same as in the case withthe composition of the present invention.

Hereinafter, the present invention will be described in more detail withreference to EXAMPLES and TEST EXAMPLES below, but is not deemed to belimited thereto.

EXAMPLES Reference Example 14-{[(2S,6R)-6-(4-Benzamido-2-oxopyrimidin-1-yl)-4-tritylmorpholin-2-yl]methoxy}-4-oxobutanoicAcid Loaded onto Aminomethyl Polystyrene Resin Step 1: Production of4-{[(2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl]methoxy}-4-oxobutanoicAcid

Under argon atmosphere, 22.0 g ofN-{1-[(2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl]-2-oxo-1,2-dihydropyrimidin-4-yl}benzamideand 7.04 g of 4-dimethylaminopyridine (4-DMAP) were suspended in 269 mLof dichloromethane, and 5.76 g of succinic anhydride was added to thesuspension, followed by stirring at room temperature for 3 hours. To thereaction solution was added 40 mL of methanol, and the mixture wasconcentrated under reduced pressure. The residue was extracted usingethyl acetate and 0.5M aqueous potassium dihydrogenphosphate solution.The resulting organic layer was washed sequentially with 0.5M aqueouspotassium dihydrogenphosphate solution, water and brine in the ordermentioned. The resulting organic layer was dried over sodium sulfate andconcentrated under reduced pressure to give 25.9 g of the product.

Step 2: Production of4-{[(2S,6R)-6-(4-benzamido-2-oxopyrimidin-1-yl)-4-tritylmorpholin-2-yl]methoxy}-4-oxobutanoicAcid Loaded onto Aminomethyl Polystyrene Resin

After 23.5 g of4-{[(2S,6R)-6-(4-benzamido-2-oxopyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl]methoxy}-4-oxobutanoicacid was dissolved in 336 mL of pyridine (dehydrated), 4.28 g of 4-DMAPand 40.3 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride were added to the solution. Then, 25.0 g of AminomethylPolystyrene Resin cross-linked with 1% DVB (manufactured by TokyoChemical Industry Co., Ltd., A1543) and 24 mL of triethylamine wereadded to the mixture, followed by shaking at room temperature for 4days. After completion of the reaction, the resin was taken out byfiltration. The resulting resin was washed sequentially with pyridine,methanol and dichloromethane in the order mentioned, and dried underreduced pressure. To the resulting resin were added 150 mL oftetrahydrofuran (dehydrate), 15 mL of acetic anhydride and 15 mL of2,6-lutidine, and the mixture was shaken at room temperature for 2hours. The resin was taken out by filtration, washed sequentially withpyridine, methanol and dichloromethane in the order mentioned, and driedunder reduced pressure to give 33.7 g of the product.

The loading amount of the product was determined by measuring UVabsorbance at 409 nm of the molar amount of the trityl per g resin usinga known method. The loading amount of the resin was 397.4 μmol/g.

Conditions of UV Measurement

-   -   Device: U-2910 (Hitachi, Ltd.)    -   Solvent: methanesulfonic acid    -   Wavelength: 265 nm    -   ε Value: 45000

Reference Example 24-Oxo-4-{[(2S,6R)-6-(6-oxo-2-[2-phenoxyacetamido]-1H-purin-9-yl)-4-tritylmorpholin-2-yl]methoxy}butanoicacid Loaded onto 2-aminomethylpolystyrene Resin Step 1: Production ofN²-(phenoxyacetyl)guanosine

Guanosine, 100 g, was dried at 80° C. under reduced pressure for 24hours. After 500 mL of pyridine (anhydrous) and 500 mL ofdichloromethane (anhydrous) were added thereto, 401 mL ofchlorotrimethylsilane was dropwise added to the mixture under an argonatmosphere at 0° C., followed by stirring at room temperature for 3hours. The mixture was again ice-cooled and 66.3 g of phenoxyacetylchloride was dropwise added thereto. Under ice cooling, the mixture wasstirred for further 3 hours. To the reaction solution was added 500 mLof methanol, and the mixture was stirred at room temperature overnight.The solvent was then removed by distillation under reduced pressure. Tothe residue was added 500 mL of methanol, and concentration underreduced pressure was performed 3 times. To the residue was added 4 L ofwater, and the mixture was stirred for an hour under ice cooling. Theprecipitates formed were taken out by filtration, washed sequentiallywith water and cold methanol and then dried to give 150.2 g of theobjective compound (yield: 102%) (cf. Org. Lett. (2004), Vol. 6, No. 15,2555-2557).

Step 2:N-{9-[(2R,6S)-6-(hydroxymethyl)-4-morpholin-2-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}-2-phenoxyacetamidep-toluenesulfonate

In 480 mL of methanol was suspended 30 g of the compound obtained inStep 1, and 130 mL of 2N hydrochloric acid was added to the suspensionunder ice cooling. Subsequently, 56.8 g of ammonium tetraboratetetrahydrate and 16.2 g of sodium periodate were added to the mixture inthe order mentioned and stirred at room temperature for 3 hours. Thereaction solution was ice cooled and the insoluble matters were removedby filtration, followed by washing with 100 mL of methanol. The filtrateand washing liquid were combined and the mixture was ice cooled. To themixture was added 11.52 g of 2-picoline borane. After stirring for 20minutes, 54.6 g of p-toluenesulfonic acid monohydrate was slowly addedto the mixture, followed by stirring at 4° C. overnight. Theprecipitates were taken out by filtration and washed with 500 mL of coldmethanol and dried to give 17.7 g of the objective compound (yield:43.3%).

¹H NMR (δ, DMSO-d6): 9.9-9.2 (2H, br), 8.35 (1H, s), 7.55 (2H, m), 7.35(2H, m), 7.10 (2H, d, J=7.82 Hz), 7.00 (3H, m), 5.95 (1H, dd, J=10.64,2.42 Hz), 4.85 (2H, s), 4.00 (1H, m), 3.90-3.60 (2H, m), 3.50-3.20 (5H,m), 2.90 (1H, m), 2.25 (3H, s)

Step 3: Production ofN-{9-[(2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}-2-phenoxyacetamide

In 30 mL of dichloromethane was suspended 2.0 g of the compound obtainedin Step 2, and 13.9 g of triethylamine and 18.3 g of trityl chloridewere added to the suspension under ice cooling. The mixture was stirredat room temperature for an hour. The reaction solution was washed withsaturated sodium bicarbonate aqueous solution and then with water, anddried. The organic layer was concentrated under reduced pressure. To theresidue was added 40 mL of 0.2M sodium citrate buffer (pH 3)/methanol(1:4 (v/v)), and the mixture was stirred. Subsequently, 40 mL of waterwas added and the mixture was stirred for an hour under ice cooling. Themixture was taken out by filtration, washed with cold methanol and driedto give 1.84 g of the objective compound (yield: 82.0%).

Step 4: Production of4-oxo-4-{[(2S,6R)-6-(6-oxo-2-[2-phenoxyacetamido]-1H-purin-9-yl)-4-tritylmorpholin-2-yl]methoxy}butanoicAcid Loaded onto Aminomethyl Polystyrene Resin

The title compound was produced in a manner similar to REFERENCE EXAMPLE1, except thatN-{9-[(2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}-2-phenoxyacetamidewas used in this step, instead ofN-{1-[(2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl]-2-oxo-1,2-dihydropyrimidin-4-yl}benzamideused in Step 1 of REFERENCE EXAMPLE 1.

Reference Example 34-{[(2S,6R)-6-(5-Methyl-2,4-dioxo-3,4-dihydropyrimidin-1-yl)-4-tritylmorpholin-2-yl]methoxy}-4-oxobutanoicAcid Loaded onto Aminomethyl Polystyrene Resin

The title compound was produced in a manner similar to REFERENCE EXAMPLE1, except that1-[(2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl]-5-methylpyrimidine-2,4(1H,3H)-dionewas used in this step, instead ofN-{1-[(2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl]-2-oxo-1,2-dihydropyrimidin-4-yl}benzamideused in Step 1 of REFERENCE EXAMPLE 1.

Reference Example 41,12-Dioxo-1-(4-tritylpiperazin-1-yl)-2,5,8,11-tetraoxa-15-pentadecanoicAcid Loaded onto Aminomethyl Polystyrene Resin

The title compound was produced in a manner similar to REFERENCE EXAMPLE1, except that 2-[2-(2-hydroxyethoxy)ethoxy]ethyl4-tritylpiperazine-1-carboxylic acid (the compound described in WO2009/064471) was used in this step, instead ofN-{1-[(2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl]-2-oxo-1,2-dihydropyrimidin-4-yl}benzamide.

According to the descriptions in EXAMPLES 1 to 12 and REFERENCE EXAMPLES1 to 3 below, various types of PMO shown by PMO Nos. 1-11 and 13-16 inTABLE 2 were synthesized. The PMO synthesized was dissolved ininjectable water (manufactured by Otsuka Pharmaceutical Factory, Inc.).PMO No. 12 was purchased from Gene Tools, LLC.

TABLE 2 Target sequence PMO in No. exon 53 Note SEQ ID NO: 1 31-55 5′end: group (3) SEQ ID NO: 4 2 32-53 5′ end: group (3) SEQ ID NO: 8 332-56 5′ end: group (3) SEQ ID NO: 11 4 33-54 5′ end: group (3) SEQ IDNO: 15 5 34-58 5′ end: group (3) SEQ ID NO: 25 6 36-53 5′ end: group (3)SEQ ID NO: 32 7 36-55 5′ end: group (3) SEQ ID NO: 34 8 36-56 5′ end:group (3) SEQ ID NO: 35 9 36-57 5′ end: group (3) SEQ ID NO: 36 10 33-575′ end: group (3) SEQ ID NO: 18 11 39-69 Sequence corresponding to SEQID NO: 38 H53A(+39 + 69) (cf. Table in 1) Non-Patent Document 3, 5′ end:group (3) 12 30-59 Sequence corresponding SEQ ID NO: 39 to h53A30/1 (cf.Table 1) in Non-Patent Document 5, 5′ end: group (2) 13 32-56 5′ end:group (1) SEQ ID NO: 11 14 36-56 5′ end: group (1) SEQ ID NO: 35 1530-59 Sequence corresponding SEQ ID NO: 39 to h53A30/1 (cf. Table 1) inNon-Patent Document 5 5′ end: group (3) 16 23-47 Sequence correspondingSEQ ID NO: 47 to SEQ ID NO: 429 described in Patent Document 4, 5′ end:group (3)

Example 1

PMO No. 8

4-{[(2S,6R)-6-(4-Benzamido-2-oxopyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl]methoxy}-4-oxobutanoicacid, loaded onto aminomethyl polystyrene resin (REFERENCE EXAMPLE 1), 2g (800 μmop was transferred to a reaction vessel, and 30 mL ofdichloromethane was added thereto. The mixture was allowed to stand for30 minutes. After the mixture was further washed twice with 30 mL ofdichloromethane, the following synthesis cycle was started. The desiredmorpholino monomer compound was added in each cycle to give thenucleotide sequence of the title compound.

TABLE 3 Step Reagent Volume (mL) Time (mm) 1 deblocking solution 30 2.02 deblocking solution 30 2.0 3 deblocking solution 30 2.0 4 deblockingsolution 30 2.0 5 deblocking solution 30 2.0 6 deblocking solution 302.0 7 neutralizing solution 30 1.5 8 neutralizing solution 30 1.5 9neutralizing solution 30 1.5 10 neutralizing solution 30 1.5 11neutralizing solution 30 1.5 12 neutralizing solution 30 1.5 13dichloromethane 30 0.5 14 dichloromethane 30 0.5 15 dichloromethane 300.5 16 coupling solution B 20 0.5 17 coupling solution A 6-11 90.0 18dichloromethane 30 0.5 19 dichloromethane 30 0.5 20 dichloromethane 300.5 21 capping solution 30 3.0 22 capping solution 30 3.0 23dichloromethane 30 0.5 24 dichloromethane 30 0.5 25 dichloromethane 300.5

The deblocking solution used was a solution obtained by dissolving amixture of trifluoroacetic acid (2 equivalents) and triethylamine (1equivalent) in a dichloromethane solution containing 1% (v/v) ethanoland 10% (v/v) 2,2,2-trifluoroethanol to be 3% (w/v). The neutralizingsolution used was a solution obtained by dissolvingN,N-diisopropylethylamine in a dichloromethane solution containing 25%(v/v) 2-propanol to be 5% (v/v). The coupling solution A used was asolution obtained by dissolving the morpholino monomer compound in1,3-dimethyl-2-imidazolidinone containing 10% (v/v)N,N-diisopropylethylamine to be 0.15M. The coupling solution B used wasa solution obtained by dissolving N,N-diisopropylethylamine in1,3-dimethyl-2-imidazolidinone to be 10% (v/v). The capping solutionused was a solution obtained by dissolving 20% (v/v) acetic anhydrideand 30% (v/v) 2,6-lutidine in dichloromethane.

The aminomethyl polystyrene resin loaded with the PMO synthesized abovewas recovered from the reaction vessel and dried at room temperature forat least 2 hours under reduced pressure. The dried PMO loaded ontoaminomethyl polystyrene resin was charged in a reaction vessel, and 200mL of 28% ammonia water-ethanol (1/4) was added thereto. The mixture wasstirred at 55° C. for 15 hours. The aminomethyl polystyrene resin wasseparated by filtration and washed with 50 mL of water-ethanol (1/4).The resulting filtrate was concentrated under reduced pressure. Theresulting residue was dissolved in 100 mL of a solvent mixture of 20 mMacetic acid-triethylamine buffer (TEAA buffer) and acetonitrile (4/1)and filtered through a membrane filter. The filtrate obtained waspurified by reversed phase HPLC. The conditions used are as follows.

TABLE 4 Column XTerra MS18 (Waters, φ50x 100 mm, 1CV = 200 mL) Flow rate60 mL/min Column temperature room temperature Solution A 20 mM TEAAbuffer Solution B CH₃CN Gradient (B) conc. 20→50%/9CV

Each fraction was analyzed and the product was recovered in 100 mL ofacetonitrile-water (1/1), to which 200 mL of ethanol was added. Themixture was concentrated under reduced pressure. Further drying underreduced pressure gave a white solid. To the resulting solid was added300 mL of 10 mM phosphoric acid aqueous solution to suspend the solid.To the suspension was added 10 mL of 2M phosphoric acid aqueoussolution, and the mixture was stirred for 15 minutes. Furthermore, 15 mLof 2M sodium hydrate aqueous solution was added for neutralization.Then, 15 mL of 2M sodium hydroxide aqueous solution was added to makethe mixture alkaline, followed by filtration through a membrane filter(0.45 μm). The mixture was thoroughly washed with 100 mL of 10 mM sodiumhydroxide aqueous solution to give the product as an aqueous solution.

The resulting aqueous solution containing the product was purified by ananionic exchange resin column. The conditions used are as follows.

TABLE 5 Column Source 30Q (GE Healthcare, φ40x 150 mm, 1CV = 200 mL)Flow rate 80 mL/min Column temp. room temperature Solution A 10 mMsodium hydroxide aqueous solution Solution B 10 mM sodium hydroxideaqueous solution, 1M sodium chloride aqueous solution Gradient (B) conc.5→35%/15CV

Each fraction was analyzed (on HPLC) and the product was obtained as anaqueous solution. To the resulting aqueous solution was added 225 mL of0.1M phosphate buffer (pH 6.0) for neutralization. The mixture wasfiltered through a membrane filter (0.45 μm). Next, ultrafiltration wasperformed under the conditions described below.

TABLE 6 Filter PELLICON2 MINI FILTER PLBC 3K Regenerated Cellulose,Screen Type C Size 0.1 m²

The filtrate was concentrated to give approximately 250 mL of an aqueoussolution. The resulting aqueous solution was filtered through a membranefilter (0.45 μm). The aqueous solution obtained was freeze-dried to give1.5 g of the objective compound as a white cotton-like solid.

ESI-TOF-MS Calcd.: 6924.82.

Found: 6923.54.

Example 2

PMO. No. 1

The title compound was produced in accordance with the procedure ofEXAMPLE 1.

MALDI-TOF-MS Calcd.: 8291.96.

Found: 8296.24.

Example 3

PMO. No. 2

The title compound was produced in accordance with the procedure ofEXAMPLE 1.

ESI-TOF-MS Calcd.: 7310.13.

Found: 7309.23.

Example 4

PMO. No. 3

The title compound was produced in accordance with the procedure ofEXAMPLE 1.

ESI-TOF-MS Calcd.: 8270.94.

Found: 8270.55.

Example 5

PMO. No. 4

The title compound was produced in accordance with the procedure ofEXAMPLE 1, except that4-(((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methoxy)-4-oxobutanoicacid (REFERENCE EXAMPLE 3) loaded onto aminomethyl polystyrene resin wasused as the starting material.

ESI-TOF-MS Calcd.: 7310.13.

Found: 7310.17.

Example 6

PMO. No. 5

The title compound was produced in accordance with the procedure ofEXAMPLE 1, except that4-(((2S,6R)-6-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-tritylmorpholin-2-yl)methoxy)-4-oxobutanoicacid loaded onto aminomethyl polystyrene resin (REFERENCE EXAMPLE 3) wasused as the starting material.

ESI-TOF-MS Calcd.: 8270.94.

Found: 8270.20.

Example 7

PMO. No. 6

The title compound was produced in accordance with the procedure ofEXAMPLE 1.

ESI-TOF-MS Calcd.: 5964.01.

Found: 5963.68.

Example 8

PMO. No. 7

The title compound was produced in accordance with the procedure ofEXAMPLE 1.

ESI-TOF-MS Calcd.: 6609.55.

Found: 6608.85.

Example 9

PMO. No. 9

The title compound was produced in accordance with the procedure ofEXAMPLE 1, except that4-oxo-4-(((2S,6R)-6-(6-oxo-2-(2-phenoxyacetamido)-1H-purin-9(6H)-yl)-4-tritylmorpholin-2-yl)methoxy)butanoicacid loaded onto aminomethyl polystyrene resin (REFERENCE EXAMPLE 2) wasused as the starting material.

ESI-TOF-MS Calcd.: 7280.11.

Found: 7279.42.

Example 10

PMO. No. 10

The title compound was produced in accordance with the procedure ofEXAMPLE 1, except that4-oxo-4-(((2S,6R)-6-(6-oxo-2-(2-phenoxyacetamido)-1H-purin-9(6H)-yl)-4-tritylmorpholin-2-yl)methoxy)butanoicacid loaded onto aminomethyl polystyrene resin (REFERENCE EXAMPLE 2) wasused as the starting material.

ESI-TOF-MS Calcd.: 8295.95.

Found: 8295.91.

Example 11

PMO. No. 13

The title compound was produced in accordance with the procedure ofEXAMPLE 1, except that1,12-dioxo-1-(4-tritylpiperazin-1-yl)-2,5,8,11-tetraoxa-15-pentadecanoicacid loaded onto aminomethyl polystyrene resin (REFERENCE EXAMPLE 4) wasused as the starting material.

ESI-TOF-MS Calcd.: 7276.15.

Found: 7276.69.

Example 12

PMO. No. 14

The title compound was produced in accordance with the procedure ofEXAMPLE 1, except that1,12-dioxo-1-(4-tritylpiperazin-1-yl)-2,5,8,11-tetraoxa-15-pentadecanoicacid loaded onto aminomethyl polystyrene resin (REFERENCE EXAMPLE 4) wasused as the starting material.

ESI-TOF-MS Calcd.: 8622.27.

Found: 8622.29.

Comparative Example 1

PMO. No. 11

The title compound was produced in accordance with the procedure ofEXAMPLE 1.

ESI-TOF-MS Calcd.: 10274.63.

Found: 10273.71.

Comparative Example 2

PMO. No. 15

The title compound was produced in accordance with the procedure ofEXAMPLE 1.

ESI-TOF-MS Calcd.: 9941.33.

Found: 9940.77.

Comparative Example 3

PMO. No. 16

The title compound was produced in accordance with the procedure ofEXAMPLE 1.

ESI-TOF-MS Calcd.: 8238.94.

Found: 8238.69.

Test Example 1

In Vitro Assay

Using an Amaxa Cell Line Nucleofector Kit L on Nucleofector II (Lonza),10 μM of the oligomers PMO Nos. 1 to 8 of the present invention and theantisense oligomer PMO No. 11 were transfected with 4×10⁵ of RD cells(human rhabdomyosarcoma cell line). The Program T-030 was used.

After transfection, the cells were cultured overnight in 2 mL of Eagle'sminimal essential medium (EMEM) (manufactured by Sigma, hereinafter thesame) containing 10% fetal calf serum (FCS) (manufactured by Invitrogen)under conditions of 37° C. and 5% CO₂. The cells were washed twice withPBS (manufactured by Nissui, hereinafter the same) and 500 μl of ISOGEN(manufactured by Nippon Gene) was added to the cells. After the cellswere allowed to stand at room temperature for a few minutes to lyse thecells, the lysate was collected in an Eppendorf tube. The total RNA wasextracted according to the protocol attached to ISOGEN. Theconcentration of the total RNA extracted was determined using a NanoDropND-1000 (manufactured by LMS).

One-Step RT-PCR was performed with 400 ng of the extracted total RNAusing a Titan One Tube RT-PCR Kit (manufactured by Roche). A reactionsolution was prepared in accordance with the protocol attached to thekit. A PTC-100 (manufactured by MJ Research) was used as a thermalcycler. The RT-PCR program used is as follows.

-   -   50° C., 30 mins: reverse transcription    -   94° C., 2 mins: thermal denaturation    -   [94° C., 10 seconds; 58° C., 30 seconds; 68° C., 45 seconds]×30        cycles: PCR amplification

68° C., 7 mins: final extension

The nucleotide sequences of the forward primer and reverse primer usedfor RT-PCR are given below.

Forward primer: 5′-AGGATTTGGAACAGAGGCGTC-3′ (SEQ ID NO: 40)

Reverse primer: 5′-GTCTGCCACTGGCGGAGGTC-3′ (SEQ ID NO: 41)

Next, a nested PCR was performed with the product amplified by RT-PCRabove using a Taq DNA Polymerase (manufactured by Roche). The PCRprogram used is as follows.

-   -   94° C., 2 mins: thermal denaturation    -   [94° C., 15 seconds; 58° C., 30 seconds; 68° C., 45 seconds]×30        cycles: PCR amplification    -   68° C., 7 mins: final extension

The nucleotide sequences of the forward primer and reverse primer usedfor the nested PCR above are given below.

Forward primer: 5′-CATCAAGCAGAAGGCAACAA-3′ (SEQ ID NO: 42)

Reverse primer: 5′-GAAGTTTCAGGGCCAAGTCA-3′ (SEQ ID NO: 43)

The reaction product, 1 μl, of the nested PCR above was analyzed using aBioanalyzer (manufactured by Agilent Technologies, Inc.).

The polynucleotide level “A” of the band with exon 53 skipping and thepolynucleotide level “B” of the band without exon 53 skipping weremeasured. Based on these measurement values of “A” and “B,” the skippingefficiency was determined by the following equation:Skipping efficiency (%)=A/(A+B)×100Experimental Results

The results are shown in FIG. 1. This experiment revealed that theoligomers PMO Nos. 1 to 8 of the present invention caused exon 53skipping with a markedly high efficiency as compared to the antisenseoligomer PMO No. 11. In particular, the oligomers PMO Nos. 3 and 8 ofthe present invention exhibited more than four times higher exonskipping efficiency than that of the antisense oligomer PMO No. 11.

Test Example 2

In Vitro Assay Using Human Fibroblasts

Human myoD gene (SEQ ID NO: 44) was introduced into TIG-119 cells (humannormal tissue-derived fibroblasts, National Institute of BiomedicalInnovation) or 5017 cells (human DMD patient-derived fibroblasts,Coriell Institute for Medical Research) using a ZsGreen1 coexpressionretroviral vector.

After incubation for 4 to 5 days, ZsGreen-positive MyoD-transformedfibroblasts were collected by FACS and plated at 5×10⁴/cm² into a12-well plate. As a growth medium, there was used 1 mL of Dulbecco'sModified Eagle Medium: Nutrient Mixture F-12 (DMEM.F-12) (InvitrogenCorp.) containing 10% FCS and 1% Penicillin/Streptomycin (P/S)(Sigma-Aldrich, Inc.).

The medium was replaced 24 hours later by differentiation medium(DMEM/F-12 containing 2% equine serum (Invitrogen Corp.), 1% P/S and ITSLiquid Media Supplement (Sigma, Inc.)). The medium was exchanged every 2to 3 days and incubation was continued for 12 to 14 days todifferentiate into myotubes.

Subsequently, the differentiation medium was replaced by adifferentiation medium containing 6 μM Endo-Porter (Gene Tools), and themorpholino oligomer was added thereto in a final concentration of 10 μM.After incubation for 48 hours, total RNA was extracted from the cellsusing a TRIzol (manufactured by Invitrogen Corp.). RT-PCR was performedwith 50 ng of the extracted total RNA using a QIAGEN OneStep RT-PCR Kit.A reaction solution was prepared in accordance with the protocolattached to the kit. An iCycler (manufactured by Bio-Rad) was used as athermal cycler. The RT-PCR program used is as follows.

-   -   50° C., 30 mins: reverse transcription    -   95° C., 15 mins: thermal denaturation    -   [94° C., 1 mins; 60° C., 1 mins; 72° C., 1 mins]×35 cycles: PCR        amplification 72° C., 7 mins: final extension

The primers used were hEX51F and hEX55R.

hEX51F: 5′-CGGGCTTGGACAGAACTTAC-3′ (SEQ ID NO: 45)

hEx55R: 5′-TCCTTACGGGTAGCATCCTG-3′ (SEQ ID NO: 46)

The reaction product of RT-PCR above was separated by 2% agarose gelelectrophoresis and gel images were captured with a GeneFlash (Syngene).The polynucleotide level “A” of the band with exon 53 skipping and thepolynucleotide level “B” of the band without exon 53 skipping weremeasured using an Image J (manufactured by National Institutes ofHealth). Based on these measurement values of “A” and “B,” the skippingefficiency was determined by the following equation.Skipping efficiency (%)=A/(A+B)×100Experimental Results

The results are shown in FIGS. 2 and 3. This experiment revealed that inTIG-119 cells, the oligomers PMO Nos. 3, 8 and 9 of the presentinvention (FIG. 2) all caused exon 53 skipping with a higher efficiencythan the antisense oligomer PMO No. 12 (FIG. 2). In particular, theoligomers PMO Nos. 3 and 8 of the present invention exhibited more thantwice higher exon skipping efficiency than that of the antisenseoligomer PMO No. 12 (FIG. 2).

Furthermore, this experiment revealed that the oligomers PMO Nos. 3 and8 to 10 of the present invention (FIG. 3) all caused exon 53 skippingwith a higher efficiency than the antisense oligomer PMO No. 12 (FIG.3). In particular, the oligomers PMO Nos. 3 and 8 of the presentinvention exhibited more than seven times higher exon skippingefficiency than that of the antisense oligomer PMO No. 12 (FIG. 3).

Test Example 3

In Vitro Assay Using Human Fibroblasts

The skin fibroblast cell line (fibroblasts from human DMD patient (exons45-52 or exons 48-52)) was established by biopsy from the medial leftupper arm of DMD patient with deletion of exons 45-52 or DMD patientwith deletion of exons 48-52. Human myoD gene (SEQ ID NO: 44) wasintroduced into the fibroblast cells using a ZsGreen1 coexpressionretroviral vector.

After incubation for 4 to 5 days, ZsGreen-positive MyoD-transformedfibroblasts were collected by FACS and plated at 5×10⁴/cm² into a12-well plate. As a growth medium, there was used 1 mL of Dulbecco'sModified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12) (InvitrogenCorp.) containing 10% FCS and 1% Penicillin/Streptomycin (P/S)(Sigma-Aldrich, Inc.).

The medium was replaced 24 hours later by a differentiation medium(DMEM/F-12 containing 2% equine serum (Invitrogen Corp.), 1% P/S and ITSLiquid Media Supplement (Sigma, Inc.)). The medium was exchanged every 2to 3 days and incubation was continued for 12, 14 or 20 days todifferentiate into myotubes.

Subsequently, the differentiation medium was replaced by adifferentiation medium containing 6 μM Endo-Porter (Gene Tools), and amorpholino oligomer was added thereto at a final concentration of 10 μM.After incubation for 48 hours, total RNA was extracted from the cellsusing a TRIzol (manufactured by Invitrogen Corp.). RT-PCR was performedwith 50 ng of the extracted total RNA using a QIAGEN OneStep RT-PCR Kit.A reaction solution was prepared in accordance with the protocolattached to the kit. An iCycler (manufactured by Bio-Rad) was used as athermal cycler. The RT-PCR program used is as follows.

-   -   50° C., 30 mins: reverse transcription    -   95° C., 15 mins: thermal denaturation    -   [94° C., 1 mins; 60° C., 1 mins; 72° C., 1 mins]×35 cycles: PCR        amplification 72° C., 7 mins: final extension

The primers used were hEx44F and h55R.

hEx44F: 5′-TGTTGAGAAATGGCGGCGT-3′ (SEQ ID NO: 48)

hEx55R: 5′-TCCTTACGGGTAGCATCCTG-3′ (SEQ ID NO: 46)

The reaction product of RT-PCR above was separated by 2% agarose gelelectrophoresis and gel images were captured with a GeneFlash (Syngene).The polynucleotide level “A” of the band with exon 53 skipping and thepolynucleotide level “B” of the band without exon 53 skipping weremeasured using an Image J (manufactured by National Institutes ofHealth). Based on these measurement values of “A” and “B,” the skippingefficiency was determined by the following equation.Skipping efficiency (%)=A/(A+B)×100Experimental Results

The results are shown in FIGS. 4 and 5. This experiment revealed thatthe oligomers PMO Nos. 3 and 8 of the present invention caused exon 53skipping with an efficiency as high as more than 80% in the cells fromDMD patient with deletion of exons 45-52 (FIG. 4) or deletion of exons48-52 (FIG. 5). Also, the oligomers PMO Nos. 3 and 8 of the presentinvention were found to cause exon 53 skipping with a higher efficiencythan that of the antisense oligomer PMO No. 15 in the cells from DMDpatient with deletion of exons 45-52 (FIG. 4).

Test Example 4

Western Blotting

The oligomer PMO No. 8 of the present invention was added to the cellsat a concentration of 10 μM, and proteins were extracted from the cellsafter 72 hours using a RIPA buffer (manufactured by Thermo FisherScientific) containing Complete Mini (manufactured by Roche AppliedScience) and quantified using a BCA protein assay kit (manufactured byThermo Fisher Scientific). The proteins were electrophoresed in NuPAGENovex Tris-Acetate Gel 3-8% (manufactured by Invitrogen) at 150V for 75minutes and transferred onto a PVDF membrane (manufactured by Millipore)using a semi-dry blotter. The PVDF membrane was blocked with a 5% ECLBlocking agent (manufactured by GE Healthcare) and the membrane was thenincubated in a solution of anti-dystrophin antibody (manufactured byNCL-Dys1, Novocastra). After further incubation in a solution ofperoxidase-conjugated goat-antimouse IgG (Model No. 170-6516, Bio-Rad),the membrane was stained with ECL Plus Western blotting system(manufactured by GE Healthcare).

Immunostaining

The oligomer PMO No. 3 or 8 of the present invention was added to thecells. The cells after 72 hours were fixed in 3% paraformaldehyde for 10minutes, followed by incubation in 10% Triton-X for 10 minutes. Afterblocking in 10% goat serum-containing PBS, the membrane was incubated ina solution of anti-dystrophin antibody (NCL-Dys1, Novocastra). Themembrane was further incubated in a solution of anti-mouse IgG antibody(manufactured by Invitrogen). The membrane was mounted with Pro LongGold Antifade reagent (manufactured by Invitrogen) and observed with afluorescence microscope.

Experimental Results

The results are shown in FIGS. 6 and 7. In this experiment it wasconfirmed by western blotting (FIG. 6) and immunostaining (FIG. 7) thatthe oligomers PMO Nos. 3 and 8 of the present invention inducedexpression of the dystrophin protein.

Test Example 5

In Vitro Assay Using Human Fibroblasts

The experiment was performed as in TEST EXAMPLE 3.

Experimental Results

The results are shown in FIG. 8. This experiment revealed that in thecells from DMD patients with deletion of exons 45-52, the oligomers PMONos. 3 to 8 of the present invention caused exon 53 skipping with ahigher efficiency than the oligomers PMO Nos. 13 and 14 of the presentinvention (FIG. 8).

Test Example 6

In Vitro Assay

Experiments were performed using the antisense oligomers of2′-O-methoxy-phosphorothioates (2-OMe-S-RNA) shown by SEQ ID NO: 49 toSEQ ID NO: 123. Various antisense oligomers used for the assay werepurchased from Japan Bio Services. The sequences of various antisenseoligomers are given below.

TABLE 7 SEQ Antisense ID oligomer Nucleotide sequence NO: H53_39-69CAUUCAACUGUUGCCUCCGGUUCUGAAGGUG 49 H53_1-25 UCCCACUGAUUCUGAAUUCUUUCAA 50H53_6-30 CUUCAUCCCACUGAUUCUGAAUUCU 51 H53_11-35UUGUACUUCAUCCCACUGAUUCUGA 52 H53_16-40 UGUUCUUGUACUUCAUCCCACUGAU 53H53_21-45 GAAGGUGUUCUUGUACUUCAUCCCA 54 H53_26-50GUUCUGAAGGUGUUCUUGUACUUCA 55 H53_31-55 CUCCGGUUCUGAAGGUGUUCUUGUA 56H53_36-60 GUUGCCUCCGGUUCUGAAGGUGUUC 57 H53_41-65CAACUGUUGCCUCCGGUUCUGAAGG 58 H53_46-70 UCAUUCAACUGUUGCCUCCGGUUCU 59H53_51-75 ACAUUUCAUUCAACUGUUGCCUCCG 60 H53_56-80CUUUAACAUUUCAUUCAACUGUUGC 61 H53_61-85 GAAUCCUUUAACAUUUCAUUCAACU 62H53_66-90 GUGUUGAAUCCUUUAACAUUUCAUU 63 H53_71-95CCAUUGUGUUGAAUCCUUUAACAUU 64 H53_76-100 UCCAGCCAUUGUGUUGAAUCCUUUA 65H53_81-105 UAGCUUCCAGCCAUUGUGUUGAAUC 66 H53_86-110UUCCUUAGCUUCCAGCCAUUGUGUU 67 H53_91-115 GCUUCUUCCUUAGCUUCCAGCCAUU 68H53_96-120 GCUCAGCUUCUUCCUUAGCUUCCAG 69 H53_101-125GACCUGCUCAGCUUCUUCCUUAGCU 70 H53_106-130 CCUAAGACCUGCUCAGCUUCUUCCU 71H53_111-135 CCUGUCCUAAGACCUGCUCAGCUUC 72 H53_116-140UCUGGCCUGUCCUAAGACCUGCUCA 73 H53_121-145 UUGGCUCUGGCCUGUCCUAAGACCU 74H53_126-150 CAAGCUUGGCUCUGGCCUGUCCUAA 75 H53_131-155UGACUCAAGCUUGGCUCUGGCCUGU 76 H53_136-160 UUCCAUGACUCAAGCUUGGCUCUGG 77H53_141-165 CCUCCUUCCAUGACUCAAGCUUGGC 78 H53_146-170GGGACCCUCCUUCCAUGACUCAAGC 79 H53_151-175 GUAUAGGGACCCUCCUUCCAUGACU 80H53_156-180 CUACUGUAUAGGGACCCUCCUUCCA 81 H53_161-185UGCAUCUACUGUAUAGGGACCCUCC 82 H53_166-190 UGGAUUGCAUCUACUGUAUAGGGAC 83H53_171-195 UCUUUUGGAUUGCAUCUACUGUAUA 84 H53_176-200GAUUUUCUUUUGGAUUGCAUCUACU 85 H53_181-205 UCUGUGAUUUUCUUUUGGAUUGCAU 86H53_186-210 UGGUUUCUGUGAUUUUCUUUUGGAU 87 H53_84-108CCUUAGCUUCCAGCCAUUGUGUUGA 88 H53_88-112 UCUUCCUUAGCUUCCAGCCAUUGUG 89H53_119-143 GGCUCUGGCCUGUCCUAAGACCUGC 90 H53_124-148AGCUUGGCUCUGGCCUGUCCUAAGA 91 H53_128-152 CUCAAGCUUGGCUCUGGCCUGUCCU 92H53_144-168 GACCCUCCUUCCAUGACUCAAGCUU 93 H53_149-173AUAGGGACCCUCCUUCCAUGACUCA 94 H53_153-177 CUGUAUAGGGACCCUCCUUCCAUGA 95H53_179-203 UGUGAUUUUCUUUUGGAUUGCAUCU 96 H53_184-208GUUUCUGUGAUUUUCUUUUGGAUUG 97 H53_188-212 CUUGGUUUCUGUGAUUUUCUUUUGG 98H53_29-53 CCGGUUCUGAAGGUGUUCUUGUACU 99 H53_30-54UCCGGUUCUGAAGGUGUUCUUGUAC 100 H53_32-56 CCUCCGGUUCUGAAGGUGUUCUUGU 101H53_33-57 GCCUCCGGUUCUGAAGGUGUUCUUG 102 H53_34-58UGCCUCCGGUUCUGAAGGUGUUCUU 103 H53_35-59 UUGCCUCCGGUUCUGAAGGUGUUCU 104H53_37-61 UGUUGCCUCCGGUUCUGAAGGUGUU 105 H53_38-62CUGUUGCCUCCGGUUCUGAAGGUGU 106 H53_39-63 ACUGUUGCCUCCGGUUCUGAAGGUG 107H53_40-64 AACUGUUGCCUCCGGUUCUGAAGGU 108 H53_32-61UGUUGCCUCCGGUUCUGAAGGUGUUCUUGU 109 H53_32-51 GGUUCUGAAGGUGUUCUUGU 110H53_35-54 UCCGGUUCUGAAGGUGUUCU 111 H53_37-56 CCUCCGGUUCUGAAGGUGUU 112H53_40-59 UUGCCUCCGGUUCUGAAGGU 113 H53_42-61 UGUUGCCUCCGGUUCUGAAG 114H53_32-49 UUCUGAAGGUGUUCUUGU 115 H53_35-52 CGGUUCUGAAGGUGUUCU 116H53_38-55 CUCCGGUUCUGAAGGUGU 117 H53_41-58 UGCCUCCGGUUCUGAAGG 118H53_44-61 UGUUGCCUCCGGUUCUGA 119 H53_35-49 UUCUGAAGGUGUUCU 120 H53_40-54UCCGGUUCUGAAGGU 121 H53_45-59 UUGCCUCCGGUUCUG 122 H53_45-62CUGUUGCCUCCGGUUCUG 123

RD cells (human rhabdomyosarcoma cell line) were plated at 3×10⁵ in a6-well plate and cultured in 2 mL of Eagle's minimal essential medium(EMEM) (manufactured by Sigma, Inc., hereinafter the same) containing10% fetal calf serum (FCS) (manufactured by Invitrogen Corp.) underconditions of 37° C. and 5% CO₂ overnight. Complexes of variousantisense oligomers (Japan Bio Services) (1 μM) for exon 53 skipping andLipofectamine 2000 (manufactured by Invitrogen Corp.) were prepared and200 μl was added to RD cells where 1.8 mL of the medium was exchanged,to reach the final concentration of 100 nM.

After completion of the addition, the cells were cultured overnight. Thecells were washed twice with PBS (manufactured by Nissui, hereafter thesame) and then 500 μl of ISOGEN (manufactured by Nippon Gene) were addedto the cells. After the cells were allowed to stand at room temperaturefor a few minutes for cell lysis, the lysate was collected in anEppendorf tube. The total RNA was extracted according to the protocolattached to ISOGEN. The concentration of the total RNA extracted wasdetermined using a NanoDrop ND-1000 (manufactured by LMS).

One-Step RT-PCR was performed with 400 ng of the extracted total RNAusing a Titan One Tube RT-PCR Kit (manufactured by Roche). A reactionsolution was prepared in accordance with the protocol attached to thekit. A PTC-100 (manufactured by MJ Research) was used as a thermalcycler. The RT-PCR program used is as follows.

-   -   50° C., 30 mins: reverse transcription    -   94° C., 2 mins: thermal denaturation    -   [94° C., 10 seconds; 58° C., 30 seconds; 68° C., 45 seconds]×30        cycles: PCR amplification    -   68° C., 7 mins: final extension

The nucleotide sequences of the forward primer and reverse primer usedfor RT-PCR are given below.

Forward primer: 5′-CATCAAGCAGAAGGCAACAA-3′ (SEQ ID NO: 42)

Reverse primer: 5′-GAAGTTTCAGGGCCAAGTCA-3′ (SEQ ID NO: 43)

Subsequently, a nested PCR was performed with the amplified product ofRT-PCR above using a Taq DNA Polymerase (manufactured by Roche). The PCRprogram used is as follows.

-   -   94° C., 2 mins: thermal denaturation    -   [94° C., 15 seconds; 58° C., 30 seconds; 68° C., 45 seconds]×30        cycles: PCR amplification    -   68° C., 7 mins: final extension

The nucleotide sequences of the forward primer and reverse primer usedfor the nested PCR above are given below.

Forward primer: 5′-AGGATTTGGAACAGAGGCGTC-3′ (SEQ ID NO: 40)

Reverse primer: 5′-GTCTGCCACTGGCGGAGGTC-3′ (SEQ ID NO: 41)

The reaction product, 1 μl, of the nested PCR above was analyzed using aBioanalyzer (manufactured by Agilent Technologies, Inc.).

The polynucleotide level “A” of the band with exon 53 skipping and thepolynucleotide level “B” of the band without exon 53 skipping weremeasured. Based on these measurement values of “A” and “B,” the skippingefficiency was determined by the following equation:Skipping efficiency (%)=A/(A+B)×100Experimental Results

The results are shown in FIGS. 9 to 17. These experiments revealed that,when the antisense oligomers were designed at exons 31-61 from the 5′end of exon 53 in the human dystrophin gene, exon 53 skipping could becaused with a high efficiency.

Test Example 7

Using an Amaxa Cell Line Nucleofector Kit L on Nucleofector II (Lonza),0.3 to 30 μM of the antisense oligomers were transfected with 3.5×10⁵ ofRD cells (human rhabdomyosarcoma cell line). The Program T-030 was used.

After the transfection, the cells were cultured overnight in 2 mL ofEagle's minimal essential medium (EMEM) (manufactured by Sigma, Inc.,hereinafter the same) containing 10% fetal calf serum (FCS)(manufactured by Invitrogen Corp.) under conditions of 37° C. and 5%CO₂. The cells were washed twice with PBS (manufactured by Nissui,hereinafter the same) and 500 μl of ISOGEN (manufactured by Nippon Gene)was then added to the cells. After the cells were allowed to stand atroom temperature for a few minutes to lyse the cells, the lysate wascollected in an Eppendorf tube. The total RNA was extracted according tothe protocol attached to ISOGEN. The concentration of the total RNAextracted was determined using a NanoDrop ND-1000 (manufactured by LMS).

One-Step RT-PCR was performed with 400 ng of the extracted total RNAusing a QIAGEN OneStep RT-PCR Kit (manufactured by Qiagen, Inc.). Areaction solution was prepared in accordance with the protocol attachedto the kit. The thermal cycler used was a PTC-100 (manufactured by MJResearch). The RT-PCR program used is as follows.

-   -   50° C., 30 mins: reverse transcription    -   95° C., 15 mins: thermal denaturation    -   [94° C., 30 seconds; 60° C., 30 seconds; 72° C., 1 mins]×35        cycles: PCR amplification    -   72° C., 10 mins: final extension

The nucleotide sequences of the forward primer and reverse primer usedfor RT-PCR are given below.

Forward primer: 5′-CATCAAGCAGAAGGCAACAA-3′ (SEQ ID NO: 42)

Reverse primer: 5′-GAAGTTTCAGGGCCAAGTCA-3′ (SEQ ID NO: 43)

The reaction product, 1 μl, of the PCR above was analyzed using aBioanalyzer (manufactured by Agilent Technologies, Inc.).

The polynucleotide level “A” of the band with exon 53 skipping and thepolynucleotide level “B” of the band without exon 53 skipping weremeasured. Based on these measurement values of “A” and “B,” the skippingefficiency was determined by the following equation:Skipping efficiency (%)=A/(A+B)×100Experimental Results

The results are shown in FIGS. 18 and 19. These experiments revealedthat the oligomer PMO No. 8 of the present invention caused exon 53skipping with a markedly high efficiency as compared to the antisenseoligomers PMO Nos. 15 and 16 (FIG. 18). It was also revealed that theoligomers PMO Nos. 3 and 8 of the present invention caused exon 53skipping with a markedly high efficiency as compared to the oligomersPMO Nos. 13 and 14 of the present invention (FIG. 19). These resultsshowed that the sequences with —OH group at the 5′ end provide a higherskipping efficiency even in the same sequences.

INDUSTRIAL APPLICABILITY

Experimental results in TEST EXAMPLES demonstrate that the oligomers ofthe present invention (PMO Nos. 1 to 10) all caused exon 53 skippingwith a markedly high efficiency under all cell environments, as comparedto the oligomers (PMO Nos. 11, 12, 15 and 16) in accordance with theprior art. The 5017 cells used in TEST EXAMPLE 2 are the cells isolatedfrom DMD patients, and the fibroblasts used in TEST EXAMPLES 3 and 5 areexon 53 skipping target cells from DMD patients. Particularly in TESTEXAMPLES 3 and 5, the oligomers of the present invention show the exon53 skipping efficiency of 90% or higher in the cells from DMD patientsthat are the target for exon 53 skipping. Consequently, the oligomers ofthe present invention can induce exon 53 skipping with a highefficiency, when DMD patients are administered.

Therefore, the oligomers of the present invention are extremely usefulfor the treatment of DMD.

Sequence Listing Free Text

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The invention claimed is:
 1. A solid-phase method of making an oligomercomprising a phosphorodiamidate morpholino oligomer (PMO) and a group atthe 5′ end of said PMO, wherein said PMO is 100% complementary to the36th to the 60th nucleotides from the 5′ end of the 53rd exon in a humandystrophin pre-mRNA, wherein the 53rd exon in said human dystrophinpre-mRNA consists of a nucleotide sequence corresponding to SEQ ID NO:1, wherein said PMO hybridizes to said human dystrophin pre-mRNA withWatson-Crick base pairing, wherein the phosphorodiamidate morpholinomonomers of said PMO have the formula:

wherein each of R² and R³ represents a methyl; wherein Base is anucleobase selected from the group consisting of: uracil, cytosine,thymine, adenine, and guanine; and wherein the group at the 5′ end ofsaid PMO has the formula:

said method comprising: a) providing Compound 1:

wherein T represents trityl, monomethoxytrityl, or dimethoxytrityl;wherein each of R² and R³ represents a methyl; and wherein B^(P) is aprotected Base, b) reacting said Compound 1 with an acid to formCompound 2;

c) reacting said Compound 2 with a morpholino monomer in the presence ofa base and a solvent; d) repeating steps b) and c) until Compound 3 iscomplete;

e) reacting said Compound 3 with a deprotecting agent to form Compound4; and

f) reacting Compound 4 with an acid to form said oligomer.
 2. The methodaccording to claim 1, wherein said acid used in step b) istrifluoroacetic acid.
 3. The method according to claim 1, wherein saidbase used in step c) is N-ethylmorpholine and said solvent isN,N-dimethylimidazolidone.
 4. The method according to claim 1, whereinsaid deprotecting agent is concentrated ammonia water used as a dilutionwith a solvent or a mixture of solvents.
 5. The method according toclaim 1, wherein said acid used in step f) is selected from phosphoricacid and hydrochloric acid.
 6. A solid-phase method of making aphosphorodiamidate morpholino oligomer (PMO) that is 100% complementaryto the 36th to the 60th nucleotides from the 5′ end of the 53rd exon ina human dystrophin pre-mRNA, wherein the 53rd exon in said humandystrophin pre-mRNA consists of a nucleotide sequence corresponding toSEQ ID NO: 1, wherein said PMO hybridizes to said human dystrophinpre-mRNA with Watson-Crick base pairing, wherein the phosphorodiamidatemorpholino monomers of said PMO have the formula:

wherein each of R² and R³ represents a methyl; wherein Base is anucleobase selected from the group consisting of: uracil, cytosine,thymine, adenine, and guanine; and wherein the 5′ end of said PMO hasthe formula:

said method comprising: a) providing Compound 1:

wherein T represents trityl, monomethoxytrityl, or dimethoxytrityl;wherein each of R² and R³ represents a methyl; and wherein B^(P) is aprotected Base; b) reacting said Compound 1 with an acid to formCompound 2;

c) reacting said Compound 2 with a morpholino monomer in the presence ofa base and a solvent; d) repeating steps b) and c) until Compound 3 iscomplete;

e) reacting said Compound 3 with a deprotecting agent to form Compound4; and

f) reacting said Compound 4 with an acid to form said PMO:


7. The method according to claim 6, wherein said acid used in step b) istrifluoroacetic acid.
 8. The method according to claim 6, wherein saidbase used in step c) is N-ethylmorpholine and said solvent isN,N-dimethylimidazolidone.
 9. The method according to claim 6, whereinsaid deprotecting agent is concentrated ammonia water used as a dilutionwith a solvent or a mixture of solvents.
 10. The method according toclaim 6, wherein said acid used in step f) is selected from phosphoricacid and hydrochloric acid.